Mast Cell Biology: Contemporary and Emerging Topics

Mast Cell Biology

ADVA V NCES IN EXPERIMENTA T L MEDICINE AND BIOLOGY Editorial Board: NATHAN A BACK, State University of New York at Buffalo IRUN U R. COHEN, The Weizmann Institute of Science ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research JOHN D. LAMBRIS, University of Pennsylvania RODOLFO PAOLETTI, University of Milan

Recent Volumes in this Series Volume 708 INVERTEBRATE IMMUNITY Kenneth Söderhäll Volume 709 HISTAMINE IN INFLAMMATION Robin L. Thurmond Volume 710 RECENT ADVANCES ON MODEL HOSTS Eleftherios Mylonakis Volume 711 EPIGENETIC CONTRIBUTIONS IN AUTOIMMUNE DISEASE Esteban Ballestar Volume 712 CYSTEINE PROTEASES OF PATHOGENIC ORGANISMS Mark W. Robinson and John P. Dalton Volume 713 CELL FUSION IN HEALTH AND DISEASE, I: CELL FUSION IN HEALTH Thomas Dittmar Volume 714 CELL FUSION IN HEALTH AND DISEASE, II: CELL FUSION IN DISEASE Thomas Dittmar Volume 715 BACTERIAL ADHESION: BIOLOGY, CHEMISTRY AND PHYSICS Dirk Linke and Adrian Goldman Volume 716 MAST CELL BIOLOGY: CONTEMPORARY AND EMERGING TOPICS       

A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher.

Mast Cell Biology Contemporary and Emerging Topics T Edited by      Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA     Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Landes Bioscience

Springer Science+Business Media, LLC Landes Bioscience Copyright ©2011 Landes Bioscience and Springer Science+Business Media, LLC All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing   # $#  %# # "     $    # $      and executed on a computer system; for exclusive use by the Purchaser of the work. Printed in the USA. Springer Science+Business Media, LLC, 233 Spring Street, New York, New York 10013, USA http://www.springer.com Please address all inquiries to the publishers: Landes Bioscience, 1806 Rio Grande, Austin, T Texas 78701, USA Phone: 512/ 637 6050; FAX: 512/ 637 6079 http://www.landesbioscience.com The chapters in this book are available in the Madame Curie Bioscience Database. http://www.landesbioscience.com/curie Mast Cell Biology: Contemporary and Emerging Topics           Landes Bioscience / Springer Science+Business Media, LLC dual imprint / Springer series: Advances in Experimental Medicine and Biology. ISBN: 978-1-4419-9532-2 &# # $#     $# ! #  $     #    $ of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data Library of Congress Cataloging-in-Publication Data

                      p. ; cm. -- (Advances in experimental medicine and biology ; v. 716) Includes bibliographical references and index. ISBN 978-1-4419-9532-2                !  "   and biology ; v. 716. [DNLM: 1. Mast Cells. W1 AD559 v.716 2011 / QS 532.5.C7] QR185.8.M35M363 2011 571.6--dc22 2011003740

DEDICATION A

The editors would like to dedicate this work to the many present and former members of the Laboratory of Allergic Diseases who have contributed to the success of the clinical and basic research programs in the LAD since the inception of the Laboratory in 1995. We feel privileged to have worked with so many talented and enthusiastic fellows, nurses, administrators and senior scientists. A special thanks to Dr. Anthony Fauci, the Director of the National Institute of Allergy and Infectious Diseases who, along with Dr. Tom T Kindt, then the Director of the NIAID Division of Intramural Research, supported the creation of the LAD; and to Dr. Katherine Zoon for her encouragement and support in the contemporary expansion of the clinical research program of the LAD. We are especially indebted to the patients who have participated in clinical research protocols directed at understanding and treating allergic diseases and systemic mast cell disorders.

v

FOREWORD

The editors of this book, Drs. Gilfillan and Metcalfe, have enlisted an outstanding group of investigators to discuss the emerging concepts in mast cell biology with respect to development of these cells, their homeostasis, their activation, as well as their roles in maintaining health on the one hand and on the other, their participation in disease. As noted by several of the contributors, there have been extraordinary advances in our understanding of these phenomena over the past 40 years since the tools became available to analyze these aspects at a molecular level. Initially, this ability was made possible by the use of so-called model systems by which phenomena such as the allergic response involving the IgE-mediated activation of mast cells, and the related basophils, could be investigated. The discovery of a rat mucosal mast cell tumor analog –the “rat basophilic leukemia” (RBL) cells—and the development of lines of IgE myeloma protein producing cell lines, permitted a rigorous analysis of the cellular receptor that triggered the explosive release of a variety of potent mediators. This was soon followed by the initial identification of the proximal post-receptor molecules that were activated when the receptor-bound IgE was aggregated by antigen or bivalent anti-IgE. As knowledge about similar systems expanded, extraordinarily powerful cell biological methods were developed. That knowledge and those methods have been productively applied to analyze those model systems and, increasingly, their normal counterparts in rodent cells and, even more recently, in human cells. At the same time, less reductive more physiological studies have vastly increased our insight into the role mast cells play in a variety of aspects of the immune response. These new insights not only expand our knowledge of cell biology in general but hopefully will be therapeutically applicable. Indeed, almost without exception, the authors of each of the articles in this compendium end their presentations by noting that their studies have helped to define new targets to which drugs could be directed in order to alleviate some of the pathological phenomena that are mediated by mast cells. However, as noted for example in the contribution by Tsai et al, the fact that mast cells have both positive and negative immunomodulatory functions present the challenge of whether such functions can be manipulated for therapeutic ends by suppressing those

vii

viii

FOREWORD

actions that result in disease without interfering with those functions that promote health by enhancing beneficial immune responses. Two recent events underscore this dilemma. On the down side is the case of Alzheimer T disease. It has long been posited that the disease is caused by the accumulation of socalled amyloid beta plaques in the brain. Because gamma secretase is believed to play an important role in formation of the plaque, a drug designed to inhibit the activity of that enzyme was thought to be promising. Indeed, a trial of such a drug, Eli Lilly’s semagacestat, had advanced to a Phase III clinical trial involving some 2600 patients. But in August of this year the multi-million dollar trial had to be abruptly halted when the developing data showed that the drug not only failed to slow progression of the disease, but was associated with a worsening of cognition and the ability to perform the tasks of daily living. On the other hand, a more hopeful conclusion is prompted by a report published that same month by the laboratory of T Tak Mak.1 That group has been studying the MAP kinase-interacting kinase 1 and 2 (Mnk1 and Mnk2) -protein serine/ threonine kinases that are activated by ERK K or p38 and which phosphorylate eIF4E, a component involved in the initiation of cap-dependent translation. Surprisingly, they found that Mnk1/2 double knockout mice not only exhibited normal cell growth and development despite an absence of eIF4E phosphorylation, but that the tumorigenesis occurring in a mouse model was suppressed by the loss of Mnk1/2. Furthermore, stable knockdown of Mnk1 in a human glioma cell line resulted in dramatically decreased tumor formation when these cells were injected into athymic nude mice. Thus, their data demonstrate an oncogenic role for Mnk1/2 in tumor development, and highlight these molecules as potential anticancer drug targets that could be inactivated with minimal side effects. Clearly, translating such findings to warrant a clinical trial will require many more steps but it shows that despite the complexity of cellular biochemical pathways it may be possible to pin-point specific components that though participating in normal functions can be dispensed with when they become involved in pathological processes. Many other results from clinical trials of rationally designed therapeutics, some disappointing, others successful such as the inhibitor of the ABL L tyrosine kinase, imatinib (Gleevec®) and the anti-tumor necrosis factor monoclonals infliximab (Remicade®) and adalimumab (Himura®), could be cited. So how realistic are the proposals that our increased insight into mast cell biology can be translated to yield therapeutic benefits? T To state my own cautious optimism in semi-quantitative terms, I believe that whereas our knowledge can be expected to continue to expand exponentially, the applicability of that knowledge will proceed only linearly and with a rather shallow slope. The enormous expense of clinical trials makes it critical that pre-clinical investigations should be exhaustive, and we must be careful not to let those who pay for our research, whether it be the general public or private investors, be encouraged to believe otherwise. Henry Metzger National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health, Bethesda, Maryland, USA  ' * <  = >   @    \ <^  <   _ (Mnk1 and Mnk2) delays tumor development. Proc Natl Acad Sci USA A 2010; 107:13964-13990.

ABOUT THE EDITORS...

ALASDAIR GILFILLAN A , PhD received a BSc from the University of Strathclyde, Glasgow, Scotland and MSc and PhD degrees in Pharmacology from the University of Manchester, England. Following completion of his PhD, he moved to the United States to undertake research in Cell Biology and Biochemistry in the Department of Pediatrics, Yale University Medical School; initially as a Postdoctoral Fellow, then as an Associate Research Scientist. From there, Dr. Gilfillan moved to F. Hoffman-La Roche Ltd (Nutley, New Jersey, USA) where he spent several years conducting preclinical research in the areas of allergy and inflammation. In 1999, he joined the Laboratory of Allergic Diseases (LAD), at the National Institute of Allergy and Infectious Diseases (Bethesda, Maryland, USA), where he currently helps direct the basic research efforts within the Mast Cell Biology Section. Dr. Gilfillan has had a long term interest in mast cell biology, and his current research focuses on the elucidation of signaling mechanisms associated with the activation of mast cells through Fc¡RI, KIT and other receptors, in health and disease. Dr. Gilfillan is the author of more than 90 scientific articles, and more than 50 abstracts. From 1994-2001, he was on the editorial board of the Journal of Pharmacology and Experimental Therapeutics. He currently serves on the editorial boards of Journal of Immunology, Immunology Letters, The Open Immunology Journal, The Open Allergy Journal, and the World Journal of Biological Chemistry.

ix

ABOUT THE EDITORS...

DEAN A METCALFE, MD, received a BS from Northern Arizona University and an MD from the University of Tennessee. T He completed a residency in Internal Medicine at the University of Michigan and received a MS in Microbiology from the University of Michigan. Dr. Metcalfe was a Fellow in Allergy and Immunology at NIH and a Fellow in Rheumatology and Immunology at the Robert Brigham Hospital and the Harvard Medical R School. In 1979, he returned to NIH as a Senior Clinical Investigator. His subsequent career has been devoted to research and public service at the NIH. There he combines clinical and basic research, patient care and administrative duties, as well as scholarly activities in the form of teaching, writing, and speaking on areas related to allergic diseases. In 1995, Dr. Metcalfe was appointed the first Chief of the newly created Laboratory of Allergic Diseases (LAD), NIAID. He is also Chief of the Mast Cell Biology Section/LAD and Associate Director of the NIH Clinical Center Allergy and Immunology Clinical Fellowship Program which is administered through the LAD. Dr. Metcalfe is an author on over 490 scientific papers and 230 abstracts; and an editor on 12 books on mast cell biology, systemic mast cell disorders, food allergy and asthma. He is a past Chairman of the American Board of Allergy and Immunology; a past President of the American Academy of Allergy, Asthma and Immunology; and a past Chairman of the ACGME-RRC for Allergy and Immunology. Dr. Metcalfe has been elected to membership in the American Clinical and Climatological Association, the Collegium Internationale Allergologicum, the American Society for Clinical Investigation, and the American Association of Physicians. xi

PARTICIPA P NTS

Soman N. Abraham Departments of Pathology, Immunology, and Molecular Genetics and Microbiology Duke University Medical Center Durham, North Carolina USA Sarah J. Austin Laboratory of Allergic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland USA Michael A. Beaven Laboratory of Molecular Immunology National Heart, Lung and Blood Institute National Institutes of Health Bethesda, Maryland USA Ulrich Blank Inserm U699, Université Paris 7 and Faculté de Médecine Paris Diderot Site Xavier Bichat Paris France

Peter Bradding Department of Respiratory Medicine   ` Leicester UK George H. Caughey Department of Medicine Pulmonary and Critical Care Division University of California at San Francisco San Francisco, California USA Enrico Crivellato Department of Medical and Morphological Research Section of Anatomy University of Udine Medical School Udine Italy Maria Ekoff Clinical Immunology and Allergy Department of Medicine Karolinska Institutet Stockholm Sweden Stephen J. Galli Department of Pathology Department of Microbiology and Immunology Stanford University School of Medicine Stanford, California USA xiii

xiv

    Laboratory of Allergic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland USA Michele Grimbaldeston Division of Human Immunology Centre for Cancer Biology and Schools of Molecular and Biomedical Sciences or Medicine University of Adelaide Adelaide Australia Michael F. Gurish Department of Medicine Division of Rheumatology, R Immunology and Allergy Harvard Medical School and Brigham and Women’s Hospital Boston, Massachusetts USA Jenny Hallgren Department of Medical Biochemistry and Microbiology Uppsala University Uppsala Sweden Laila Karra Department of Pharmacology and Experimental Therapeutics Institute for Drug Research School of Pharmacy The Hebrew University of Jerusalem Jerusalem Israel Jun-ichi Kashiwakura Division of Cell Biology La Jolla Institute for Allergy and Immunology La Jolla, California USA

PARTICIPA P NTS

Toshiaki Kawakami T Division of Cell Biology La Jolla Institute for Allergy and Immunology La Jolla, California USA Francesca Levi-Schaffer Department of Pharmacology and Experimental Therapeutics Institute for Drug Research School of Pharmacy The Hebrew University of Jerusalem Jerusalem Israel Diane S. Lidke Department of Pathology University of New Mexico Albuquerque, New Mexico USA Hong-Tao T Ma Laboratory of Molecular Immunology National Heart, Lung and Blood Institute National Institutes of Health Bethesda, Maryland USA Dean D. Metcalfe Laboratory of Allergic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland USA Henry Metzger National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health Bethesda, Maryland USA Elena P. Moiseeva Department of Infection, Immunity  { Institute for Lung Health University of Leicester Leicester UK

PARTICIPA P NTS

xv

Gunnar Nilsson Clinical Immunology and Allergy Department of Medicine Karolinska Institutet Stockholm Sweden

Domenico Ribatti Department of Human Anatomy and Histology University of Bari Medical School Bari Italy

Janet M. Oliver Department of Pathology University of New Mexico Albuquerque, New Mexico USA

Juan Rivera Laboratory of Molecular Immunogenetics National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health Bethesda, Maryland USA

Ana Olivera Laboratory of Molecular Immunogenetics National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health Bethesda, Maryland USA Iris M. Otani Division of Cell Biology La Jolla Institute for Allergy and Immunology La Jolla, California USA

Christopher P. Shelburne Department of Pathology Duke University Medical Center Durham, North Carolina USA Mindy Tsai Department of Pathology Stanford University School of Medicine Stanford, California USA Bridget S. Wilson Department of Pathology University of New Mexico Albuquerque, New Mexico USA

CONTENTS

SECTION I: INTRODUCTION 1. MAST CELL L BIOLOGY: INTRODUCTION AND OVERVIEW ......................2      # > $     Abstract......................................................................................................................................... 2 Introduction.................................................................................................................................. 2

  ! "#  "" ......................................................................... 3 Mast Cell Mediators and Disease ............................................................................................... 4 Mast Cell Activation .................................................................................................................... 5 Mast Cell Signaling ...................................................................................................................... 6 Conclusion .................................................................................................................................... 9

SECTION II: MAST CELL DEVELOPMENT AND HOMEOSTA T SIS 2. MAST CELL PROGENITOR TRAFF R ICKING AND MATUR A RATION ............14 Jenny Hallgren and Michael F. Gurish Abstract....................................................................................................................................... 14 Introduction................................................................................................................................ 14 Mast Cell Development.............................................................................................................. 15 Basal Homing of Mast Cell Progenitors................................................................................... 17 Recruitment of Mast Cell Progenitors ..................................................................................... 20 Mast Cell Hyperplasia ............................................................................................................... 24 Conclusion and Future Prospects............................................................................................. 25

xvii

xviii

CONTENTS

3. MONOMERIC Ig$ % & $  $'$( $%& )*'+' ' AND FUNCTION............................................................................................29 Jun-ichi Kashiwakura, Iris M. Otani and Toshiaki T Kawakami Abstract....................................................................................................................................... 29 Introduction................................................................................................................................ 29 IgE Ag Stimulation of Mast Cells............................................................................................ 30 IgE(-Ag) Stimulation of Mast Cells: Monomeric IgE Affects Mast Cell Function   8   .................................................................................................. 31

# +$ 8; <=; ;> +$ " ; ;> +$? ............. 33 +$<@? #J =;Q #   ................................................. 35 Signal Transduction T Induced by Monomeric IgE................................................................... 37 In Vivo V Effects of IgE(-Ag) Stimulation................................................................................... 39 Monomeric IgE and Mast Cell Differentiation ....................................................................... 40 Polyclonal IgE and Mast Cell Activation ................................................................................. 41 Conclusion .................................................................................................................................. 42

4. MAST CELL L APOPTOSIS AND SURVIVA V L......................................................47 Maria Ekoff and Gunnar Nilsson Abstract....................................................................................................................................... 47 Introduction................................................................................................................................ 47 Mast Cell Biology ....................................................................................................................... 48 Regulation of Apoptosis and Survival ...................................................................................... 49 Mast Cell Apoptosis ................................................................................................................... 52 Mast Cell Survival...................................................................................................................... 54 Mastocytosis................................................................................................................................ 55 Conclusion .................................................................................................................................. 55

SECTION III: MAST CELL ACTIVA VATION 5. REGULATORS A OF Ca2 SIGNALING IN MAST CELLS: POTENTIAL TARGETS FOR TREAT A MENT OF F MAST CELL-RELATED A DISEASES? ..................................................62 Hong-Tao T Ma and Michael A. Beaven Abstract....................................................................................................................................... 62 Introduction................................................................................................................................ 63 Early and Recent Observations on Calcium Homeostasis in Mast Cells: & )" + ....................................................................................................... 65 +   #  "  +3*   \ <\? * ................. 66 IP3-Sensitive Intracellular Ca2 @    ($  ........................... 68

#  2 $; <($?  $]   # #8.................. 71

  #  ............................................................................ 75 &   #   ^ _   ................. 76 # 8J ($ +8 $#   & Agents.................................................................................................................................. 77 Disease Correlates ...................................................................................................................... 80 Conclusion .................................................................................................................................. 80

CONTENTS

xix

6. SPA PATIO-TEMPORA R L SIGNALING IN MAST CELLS ....................................91 Bridget S. Wilson, Janet M. Oliver and Diane S. Lidke Abstract....................................................................................................................................... 91 Introduction................................................................................................................................ 91

# *  +............................................................................................. 92 Electron Microscopy Provides a Bird’s Eye V View of Fc¡*+  .................... 93 *   "   ^#  .................................. 94     ^    + %!> .................................. 94   &> !  & (8"   ^¡RI.............................. 95

  +  (  * *  ...................................................... 97 +##8Q  > ^C¡RI is Dependent on Dose and Antigen Valency V ............ 97 & +`    ;> ............................................................................. 99 + @ + "   #  8Q  J &   &  .......................................................... 100 h 8 j ................................................................................................................. 100 Conclusion and Future Directions.......................................................................................... 103

7. THE MECHANISMS OF EXOCYTOSIS IN MAST CELLS..........................107 Ulrich Blank Abstract..................................................................................................................................... 107 Introduction.............................................................................................................................. 107 Regulated Exocytosis in Mast Cells........................................................................................ 108     ................................................................................... 109 &  ; ;    ...................................................................... 109 Accessory Proteins in Mast Cell Membrane Fusion ..............................................................111    ^ ;   &  $]; & in Mast Cells ..................................................................................................................... 116 Targets of Calcium ................................................................................................................... 117 T Conclusion ................................................................................................................................ 119

8. AN EMERGING ROLE FOR THE LIPID MEDIATOR A SPHINGOSINE-1-PHOSPHATE A IN MAST CELL EFFECTOR R FUNCTION AND ALLERGIC DISEASE...........................123 Ana Olivera and Juan Rivera Abstract..................................................................................................................................... 123 Introduction.............................................................................................................................. 124 S1P P and Its Targets T ................................................................................................................... 124 Regulation and Function of S1P P in Tissues and Circulatory Fluids.................................... 124 & >kqkq* ]    * .............................................................................. 126 S1P P is Generated by Activated Mast Cells............................................................................. 127 Fc¡RI Induces S1p Formation and Export............................................................................ 131 Functions of S1p Receptors in Mast Cell Responses ............................................................ 132 q@  *     ;  *" ...................... 134 *"; # ;]  "; * 8; S1p from a Non-Mast Cell Source........................................................................................................................ 136 Conclusion ................................................................................................................................ 138

xx

CONTENTS

9. DOWN-REGULATION A OF MAST CELL RESPONSES THROUGH ITIM CONTA T INING INHIBITORY RECEPTORS.................................143 Laila Karra and Francesca Levi-Schaffer Abstract..................................................................................................................................... 143 Introduction.............................................................................................................................. 143 Cell Activation .......................................................................................................................... 144  +8k&#  & +## *.............................................................. 144 Activation and Down-Regulation of Mast Cell Responses: General................................... 146 +8; *J +TIM +TAM T i and ITSM.................................................................... 147

 @ +8; *........................................................................... 149 +8; *  & &j & .......................................................................... 155 Conclusion ................................................................................................................................ 156

SECTION IV: MAST CELLS IN HEALTH AND DISEASE 10. THE MAST CELL IN INNATE A AND ADAPTIVE IMMUNITY ..................162 Christopher P. Shelburne and Soman N. Abraham Abstract..................................................................................................................................... 162 Introduction.............................................................................................................................. 162 &  Q     .......................................................................... 163 &   8 ; ;................................................................................ 164 & @+`##;      .................................................................. 169 Mast Cell Modulation of Innate Responses to Infection ...................................................... 170

    _ +##;  + ......................................... 172 Conclusion ................................................................................................................................ 176

qq & $ % +

)%(*$ )&+(%k  IMMUNOMODULATION A ..........................................................................186 Mindy Tsai, Michele Grimbaldeston and Stephen J. Galli Abstract..................................................................................................................................... 186 Introduction.............................................................................................................................. 186 Mouse Models of Mast-Cell Function .................................................................................... 189 Mast Cell Activation ................................................................................................................ 191 Immunomodulatory Effects on Dendritic Cells .................................................................... 193 +###; $  ;#; ....................................................................... 194 Immunomodulatory Functions in Vivo V .................................................................................. 197 ( %" +###; ^   ............................................. 203 Conclusion ................................................................................................................................ 204

CONTENTS

xxi

12. MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY A MEDIATORS A ....................................................212 George H. Caughey Abstract..................................................................................................................................... 212 Introduction.............................................................................................................................. 213 "  @+`##; $............................................................................. 213 @+`##; *     ................................................................. 224 Conclusion ................................................................................................................................ 227

13. MAST CELLS IN LUNG INFLAMMATION A .................................................235 Elena P. Moiseeva and Peter Bradding Abstract..................................................................................................................................... 235 Introduction.............................................................................................................................. 235

  *#   ........................................................................................ 236

   =;  .................................................................................................... 237 Mast Cells in Lung Defense..................................................................................................... 245

      ...................................................................................... 247 Conclusion ................................................................................................................................ 260

qz & $ % +( $%$+ % %$*.........................................270 Domenico Ribatti and Enrico Crivellato Abstract..................................................................................................................................... 270 Introduction.............................................................................................................................. 270 Mast Cells in Experimental Carcinogenesis .......................................................................... 271 Mast Cell Recruitment to Tumor T ............................................................................................ 272 Mast Cells and Immune Tolerance T ......................................................................................... 273

   &# !  =# & ........................................................................... 274  !  ..................................................................................................... 275 Angiogenesis Factors Stored in Mast Cells............................................................................ 277 Mast Cells in Experimental T Tumor Angiogenesis ................................................................. 278 Mast Cells in Human T Tumor Angiogenesis ............................................................................ 278 Drugs Affecting Mast Cell Number........................................................................................ 281 Conclusion ................................................................................................................................ 281

INDEX........................................................................................................................289

ACKNOWLEDGEMENTS

We thank the chapter authors for their dedication in contributing up to date, outstanding manuscripts for Mast Cell Biology: Contemporary and Emerging Topics.

xxiii

SECTION I

INTRODUCTION

CHAPTER R1 MAST CELL BIOLOGY: Introduction and Overview    |  # > $     Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA               

Abstract:

    #      # " %  # $  of atopic disorders and anaphylaxis, on which it has been historically focused. The biochemical and signaling events responsible for the development and regulation of mast cells has been increasingly studied, aided in large part by novel breakthroughs in laboratory techniques used to study these cells. The result of these studies has      #!      # $  #  their overall biology as well as the various disease states that can now be traced to defects in mast cells. This introductory chapter outlines and highlights the various topics of mast cell biology that will be discussed in further detail in subsequent chapters.

INTRODUCTION Mast cells are cells of hematopoietic origin which have gained notoriety over the years for their role as central players in atopic disorders and anaphylaxis. Indeed, it has   # " # $#  #  #  #  #  $    recently that their role in other aspects of health and disease has been fully appreciated. The manifestations of mast cell-driven disease are considered to be a consequence of an inappropriate activation of mast cell immune responses which have evolved to protect the body against a host of pathogens and perhaps toxins. The biochemical processes regulating mast cell development and mast cell activation have been extensively investigated and comprehensively reviewed in recent years. Hence, rather than reviewing these topics at length, in this work we have opted to focus on the emerging concepts in mast cell biology

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 2

MAST CELL BIOLOGY: INTRODUCTION AND OVERVIEW

3

with regards not only to mast cell development and activation, but also on the newly        ##   * # #  % #!   $   } "   #      %#   $ on these topics. The scope of this effort cannot be all encompassing and accordingly,     $  #        !  *# <      $ #$    # #         To set the stage for the more in depth discussions that follow, we begin by presenting a brief overview of mast cell biology in general, in which we indicate those topics that will be elaborated upon in subsequent chapters.

& $ *(h&= $'$( $%& % )*'+' Mast cells, at least in the human, develop from CD34 /CD117 pluripotent progenitor cells originating in the bone marrow.1 The progression of these cells to fully mature mast cells is dependent on KIT activation which occurs as a consequence of stem cell factor (SCF)-induced KIT dimerization and auto-phosphorylation. Hence, KitW/W-v and KitW-sh/W-sh mice in which surface expression of KIT, or KIT catalytic activity, is defective, have substantially reduced mast cell numbers.2 Nevertheless, whereas human mast cells in culture require SCF for growth, mouse mast cell growth and expansion from bone marrow progenitors can be maintained by IL-3 in the absence of SCF.1 In both the mouse and human, committed bone marrow mast cell progenitors are released into the bloodstream from where they subsequently migrate into the peripheral tissues, during which time they mature and become terminally differentiated under # {$  < %# # $ $ $3 As discussed by Jenny Hallgren and Michael F Gurish in Chapter 2,4 the migration of mast cell progenitors         $     *#    " #    <        #     $  dendritic cells expressing the transcriptional regulatory protein, T-bet, requires that _4`7 integrin and the chemokine receptor, CXCR2, be expressed on the surface of the mast cell progenitors and that MAdCAM-1 and VCAM-1 be expressed on the intestinal endothelium. In contrast, the marked recruitment of committed mast cell     # $  ! %# #   $    { requires the expression of the _4`7 and _4`1 integrins on the mast cell progenitors and associated expression of VCAM-1 on the endothelium, as regulated by CXCR2. Full recruitment however requires activation of CCR2 pathways following binding of CCL2. Such migration of mast cell progenitors from the vasculature into the peripheral $    $ {   #$  !  "  #     $   !    {4 Terminally differentiated, tissue-resident mast cells are long lived, a feature, at least in the human, that is dependent upon the continued presence of SCF. Accordingly, inhibition of KIT catalytic activity by tyrosine kinase inhibitors induces human mast cell apoptosis.5 As discussed by Maria Ekoff and Gunnar Nilsson in Chapter 4,6 the Bcl-2 family of proteins are key regulators of such mast cell homeostasis through balancing mast cell survival and apoptosis. They further explain that apoptosis is regulated by both extrinsic pathways and intrinsic pathways which respond to stress from SCF deprivation, DNA damage and other intracellular stimuli and that these pathways involve caspase activation. Extrinsic apoptotic signals are transmitted through death receptors belonging

4

MAST CELL BIOLOGY

 # *~   $#  @€‚  *‚ƒ %## #!    on both murine and human mast cells. However, whereas Fas/CD95R R activation has been found to induce apoptosis in murine mast cells, in human mast cells, apoptosis follows TRAIL-R R crosslinking.6 Ekoff and Nilsson6 moreover discuss how SCF regulates mast cell survival through inactivation of FOXO3a and subsequent down-regulation of the pro-apoptotic BH3-only protein, Bim, by its ubiquitination and proteasomal degradation following its ##  *#   # $ $ †^_  ^  mice and cells have revealed that the BH3-only protein, Puma, is also critical for the induction of mast cell apoptosis following cytokine deprivation. Under such cytokine deprived-conditions, in wild type cells, both Bim and Puma are upregulated with resulting mast cell apoptosis. The role of the downstream effector proteins Bax and Bak is demonstrated by the fact that their absence abolishes most apoptotic responses induced by BH3-only proteins and by the increased mast cell numbers found in tissues of mice lacking Bax or Bak.6

MAST CELL MEDIATORS AND DISEASE The ability of mast cells to function in both health and disease is dependent on not only the relative abundance of mast cells in tissues, but also on the extent and nature of mediators released. These latter features are respectively dictated by the threshold and $    !   # ! !     #%   #   !$   {   

  #! #     $$   ^{   3 The immediate response upon mast cell activation to an appropriate stimulus is degranulation; a process characterized by the extrusion of cytoplasmic granule contents into the extracellular space by the process of exocytosis. Although the membrane-proximal signaling processes that allow mast cells to degranulate have been extensively studied and described,7,8 it is only recently that the detailed mechanics of degranulation and the molecular players regulating this process have been investigated. In Chapter 7, Ulrich Blank9 $  % <    #       %##   in the regulation of membrane fusion during exocytosis and the connection of the fusion machinery with early signaling events initiated upon mast cell activation. Mast cell granules are rich in bioactive amines, proteoglycans and proteases.3 Clinically, the most important bioactive amine present in the granules is histamine. The role of histamine in human disease is well established. For example, histamine, along with PGD2 and LTC4, induces bronchoconstriction, mucus secretion and mucosal edema and thus contributes to the symptoms of asthma.10,11 As described by Elena Moiseeva and Peter Bradding in Chapter 13,12 the relative rate of mediator release from human lung mast cells in vitro is histamine PGD2LTC4 with one-half maximal release occurring at 2, 5 and 10 minutes respectively and this appears to parallel in vivo release in the human lung. Serotonin (5-hydroxytryptamine) is also relatively abundant in mouse mast cell granules, but it only represents a minor component of the human mast cell granules13 and to date has not been shown to be a relevant mediator in human mast cell-driven disease. Mast cell granules are particularly rich in proteases. These proteases are the major proteins present in mast cells; representing up to 50% of the total protein content of the granule.14 The major protease present in human mast cells is `-tryptase.15 Chymase is also

MAST CELL BIOLOGY: INTRODUCTION AND OVERVIEW

5

present in substantial amounts in particular sub-populations of mast cells and the relative $     #  #$   #      particular mast cell phenotypes.16 These granule-associated proteases are complexed to   ˆ "  #  %## #  } #    {$ function.14 As discussed by George Caughey in Chapter 12,17 mast cell proteases have #        #  #$#   #!    {    $ #  %#   { #    !  ! ^{     !    $  #     ^ ! {  mediators.18 Both membrane-associated phospholipids and sphingolipids provide substrate $     !  ^{    *# $# #    phospholipase (cPL)A2, membrane phospholipids, particularly arachidonyl-containing phosphatidylcholine and, to a lesser extent, phosphatidylethanolamine, are hydrolyzed at the sn2 position to yield free arachidonic acid which can be subsequently metabolized by the respective actions of cyclo-oxygenase and 5-lipoxygenase to produce the eicosanoids, prostaglandin (PG)D2 and leukotriene (LT)C4.19 PLA2 activation also induces deacylation of the membrane-associated phospholipid, alkylacylglycerophosphorylcholine to form lyso-platelet activating factor (lyso-PAF) which is subsequently reacetylated by the action of acetylCoA:lyso-PAF acetyltransferase to produce PAF which has been implicated in systemic anaphylaxis.20 As discussed by Ana Olivera and Juan Rivera in Chapter 8,21 the sphingolipid-derived mediator sphingosine 1-phosphate, generated by mast cells following activation, is capable of inducing biological responses in mast cells and surrounding tissues. It, thus, may play a role in the regulation of diverse biological systems, including the immune system. Such responses can be mediated by both receptor-mediated and receptor-independent pathways.22 Following these initial events, which happen within minutes of mast cell activation, there occurs an enhancement of gene expression leading to the generation of an array of cytokines including GM-CSF, TNF_, IL-3, IL-4, IL-5, IL-6, IL-10 and IL-13, chemokines including CCL2, CCL3, CCL5 and CXCL8 and growth factors such as SCF, FGF, VEGF and angiogenin.23-29 As discussed by Christopher Shelburne and Soman Abraham in Chapter 10,30 by Mindy Tsai, Michele Grimbaldeston, and Stephen Galli in Chapter 11,2 by Elena Moiseeva and Peter Bradding in Chapter 13,12 and by Domenico Ribatti and Enrico Crivellato in Chapter 14,31 #    #!   $ {$  $ $ tissues and can result in a wide array of cellular responses, including induction of immune  {   $ #    $ 

MAST CELL ACTIVATION Mast cells express a multiplicity of cell surface receptors which have the capacity to impact mast cell responses through the regulation of proliferation, migration and activation. However, the two major receptors responsible for regulating mast cell functions are KIT  # ##     = ¡RI.32,33 Allergen-induced mast cell activation occurs as a consequence of Fc¡RI aggregation # $# ^  ^<  ^  = $ $  # Fc¡RI.33 However, as described by Jun-ichi Kashiwakura, Iris M. Otani and Toshiaki Kawakami in Chapter 3,34 recent studies have provided evidence that occupancy of Fc¡RI with IgE, in the absence of antigen, will provoke mast cell responses. These responses,

6

MAST CELL BIOLOGY

which are attributed to “monomeric” IgE, include induction of chemokine production, mast cell chemotaxis and the prevention of apoptosis.35 Regardless, it is clear that Fc¡RI aggregation is required to elicit the necessary signaling events for the full extent and   ^$ {   36 There is an increasing appreciation that such mast cell activation can be up-regulated or down-regulated through other receptors expressed on mast cells and that many of these receptors can by themselves activate mast cells.37,38 However, the role of these receptors in regulating mast cell function in a physiological setting remains unclear. As described elsewhere in this overview, KIT is an essential growth and anti-apoptotic factor for the generation of mast cells from progenitors and for the subsequent survival of mature, tissue-resident mast cells. SCF-dependent KIT activation, however, also enhances antigen-mediated mast cell degranulation and cytokine production.39,40 Similarly, signaling events initiated by KIT and Fc¡RI synergistically interact to markedly enhance mast cell chemotaxis.41 These responses are also observed with a number of GPCR R agonists including PGE2 and adenosine.41 The demonstration that a wide array of bacterial products, viral products, parasite products, toxins, Toll-Like Receptor (TLR) agonists and host defense products also induce activation of mast cells has led to the realization that mast cells are an important component of innate immunity as outlined by Christopher Shelburne and Soman Abraham in Chapter 10.30 The consensus of data, however, suggests that unlike antigen-, KIT- and GPCR-mediated responses, those elicited through TLRs interacting with viral, bacterial and parasitic products, are likely restricted to induction of cytokine and chemokine production. As with KIT and GPCRs, such responses are synergistically enhanced when mast cells are co-activated through the TLRs and Fc¡RI.42 This differential regulation of          !       { # ! $}     $     #  unlike the transduction pathways induced by Fc¡RI, GPCRs and KIT, signals relayed by the major adaptor molecule for the TLRs, MyD88, do not lead to enhanced intracellular calcium levels, a necessary signal for mast cell degranulation.42 It is of interest that another MyD88-linked receptor, the IL-33 receptor, enhances cytokine production in mouse and human mast cells,43 while having minimal effect on degranulation.44

MAST CELL SIGNALING    #  }  # $   #  pathways that regulate mast cell responses has been an active area of research over the past two decades and much is now known regarding these processes. These events have been extensively reviewed in a series of articles to which the readers are referred to for further details.7,8,33,37,45 Here we only present a brief overview of this topic, primarily focusing on Fc¡RI. Signaling initiated by Fc¡RI aggregation leads to the intracellular assembly of a membrane-associated receptor-signaling complex. Fc¡RI comprises an IgE-binding _ subunit, a tetramembrane-spanning ` subunit and homodimeric disulphide-linked a chain tandem subunit.33 Both the ` and a chains contribute to the transfer of signals across the membrane. However, whereas the a chain is essential for transducing signals required for mast cell activation, the role of the ` chain appears to be to amplify the signals induced by the a chains.33

MAST CELL BIOLOGY: INTRODUCTION AND OVERVIEW

7

Although it is clear that the subsequent receptor-proximal signaling events involve a series of protein phosphorylation events and protein-protein interactions,7 the precise locations where interactions take place in the context of the three dimensional structure of the cell and the real time kinetics of these processes, are relatively unknown. However, as described by Bridget Wilson, Janet Oliver and Diane Lidke in Chapter 6,46 a concept that has emerged during the past decade centers on the localization of early signal transduction events to specialized membrane microdomains. As described, electron microscopy and other approaches have revealed that, following aggregation, Fc¡‚     $ }      ‰#Š %## may represent morphological evidence of lipid rafts. These rafts, otherwise termed glycolipid-enriched domains, have been described based on their physical properties and chemical composition.47 However, these studies also reveal that these regions are not uniform and that not all signaling molecules localize to the same domains.48 It thus remains to be determined how the signaling molecules restricted to the discretely separate membrane domains interact. Nevertheless, biochemical approaches have provided evidence that, following antigen challenge, aggregated Fc¡RI coalesce with the putative lipid rafts, where they interact with key signaling molecules to initiate the cascades required for downstream mediator release.49,50 The initial key receptor-signaling protein interactions required for mediator release follow phosphorylation of immunoglobulin receptor activation motifs (ITAMs) contained within the ` and a chain cytosolic domains as mediated by the Src family tyrosine kinase Lyn; and subsequent recruitment, phosphorylation and activation of Syk tyrosine kinase. There still remains an enigma, however, regarding the role of Lyn in these initial steps. Although it is assumed that Lyn is responsible for the initial phosphorylation of the Fc¡RI ` and a chain ITAMs and thus would be essential for downstream mediator release, data from lyn
8

MAST CELL BIOLOGY

functionally related PLC` through GPCR `a subunits. TLRs, however, activate neither PLCa nor PLC`, thus explaining their lack of effect on mast cell degranulation. Through the hydrolysis of phosphoinositide 4,5, bisphosphate (PIP2) and the consequential production of inositol trisphosphate (IP3) and diacylglycerol, PLC activation leads respectively to an increase in cytosolic calcium levels and activation of protein kinase C (PKC).60 IP3 induces elevated cytosolic calcium concentrations by receptor-mediated liberation of calcium from the endoplasmic reticulum (ER).61 The emptying of the ER stores of calcium in this manner triggers a secondary, more pronounced, calcium signal through store operated calcium entry (SOCE) from extracellular sources. As described by Hong-Tao Ma and Michael Beaven in Chapter 5,62 recent studies have begun to identify the molecular players and interactions that regulate this latter process. In this respect, the  #  #   $   # =‚ #       interacting molecule-1 (STIM1)63,64 and the corresponding calcium channel on the cell membrane allowing SOCE as ORAI1.65,66 Other calcium channels, termed transient receptor potential canonical (TRPC) channels, also likely contribute to SOCE. However, the precise manner in which TRPC channels interact with ORAI and STIM has yet to be determined. The calcium signal is eventually terminated upon re-uptake of calcium and replenishment of the calcium stores within the ER R via an ATP-dependent sarco/ER Ca2 ATPase (SERCA) pump; and removal of excess cytosolic calcium across the cell membrane by TRPMV4-mediated depolarization of the cell membrane, through Na /Ca2

exchange, or through ATP-dependent plasma membrane Ca2 ATPase (PMCA) pump.67 PI3K K phosphorylates PIP2 at the 3v position, thereby generating phosphoinositide 3,4,5 trisphosphate (PIP3).68 This provides membrane docking sites for PH domain-containing signaling proteins, for example, PLCa, Btk, PDK1 and AKT.68 PI3K K is a family of heterodimeric complexes comprising a catalytic and an adaptor subunit. Both KIT and the Fc¡RI signal through the PI3Kb family member,69 whereas GPCRs signal through the PI3K Ka family member.70 PI3K K is indispensible for KIT mediated mast cell responses and for Fc¡‚^ <  $ *# < { #    $ downstream signaling cascades by PI3K, including those regulated by the MAP kinases, ERK1/2, p36 and JN J K, mTORC1 and mTORC2, and GSK3`, which contribute to the regulation of gene transcription and or chemotactic responses.70 PI3K K also contributes to mast cell degranulation and the enhancement of this response by KIT. However, a fraction of Fc¡‚^  $      # {$  \Œ71 This has led to the suggestion that PI3K K contributes to degranulation through the recruitment  †<  $‹$    \ƒ@a-mediated calcium signals required for degranulation.72,37 The status of mast cell activation at any point in time is in reality a balance between the signaling pathways discussed above and those that down-regulate these processes. As discussed by Laila Karra and Francesca Levi-Schaffer in Chapter 9,73 #    essential to avoid inappropriate or exaggerated mast cell-mediated responses. A variety of receptors are expressed on mast cells that posses the capacity to downregulate and thus buffer, receptor-induced mast cell activation.74 These receptors are characterized by a conserved immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytosolic domains which, when phosphorylated, recruits the protein phosphatase SHP-1 and the lipid phosphatases SHIP1 and SHIP2. Whereas SHP-1 reverses protein-protein interactions mediated by tyrosine kinases, SHIP1 and 2 dephosphorylate PIP3, thus reversing the association of essential PH domain-containing signaling proteins with the membrane-associated signaling complex.74 As discussed,73 disruption of the balance

MAST CELL BIOLOGY: INTRODUCTION AND OVERVIEW

9

between these inhibitory pathways and those that lead to up-regulation of mast cell responses, could result in inappropriate activation of mast cells leading to disease conditions.

CONCLUSION The understanding of mast cell biology has dramatically increased over the past two    $  #  ‘ ! *#   # % # !  of growth factors in both mouse and man that allowed the culture of mast cells from marrow and peripheral blood. The second major breakthrough was the realization that mast cells could be cultured from the marrow of mice with either known genetic defects   %#     "  %   $     % # development of approaches where mast cells could be used to replete the mast cell          *#   # % #  of the role of mast cells in diseases states, at least in the mouse. Finally, the application of genomics and proteomics applied to the study of human mast cells has allowed the association of mast cell defects to human disease states. One major example of this was the association of mutations in KIT with human mastocytosis. *# % < $ }  # #  %## { #  $  # individual chapter authors, well illustrates how mast cell biology has advanced and how these discoveries have facilitated the recognition and acceptance of mast cells as critical to both innate and acquired human immune responses.

ACKNOWLEDGEMENT Research in the authors’ laboratory is supported by the Intramural Research Program of NIAID, National Institutes of Health.

REFERENCES 1. Kirshenbaum AS, Goff JP, Semere T et al. Demonstration that human mast cells arise from a progenitor cell population that is CD34 , c-kit and expresses aminopeptidase N (CD13). Blood 1999; 94(7):2333-2342. _ *     >    $ $$$      Metcalfe DD, eds. Mast Cell Biology: Contemporary and Emerging Topics. Austin/New York: Landes Bioscience/Springer Science Business Media; 2011:186-211. 3. Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev 1997; 77(4):1033-1079. ’ `  > $ #        <  $         eds. Mast Cell Biology: Contemporary and Emerging Topics. Austin/New York: Landes Bioscience/ Springer Science Business Media; 2011:14-28.  > †  < @     \#     # *  %#     # $ consideration for mast cell disorders. Br J Pharmacol 2008; 154(8):1572-1582. “ =<  ~      $ !          @ † Contemporary and Emerging Topics. Austin/New York: Landes Bioscience/Springer Science Business Media; 2011:47-60. ”     ‚!  > *#   < % < $   ! $ ‚! _••€– 228(1):149-169. — ‚!  >     $ $    ! >   @ $ _••“– 117(6):1214-1225; quiz 1226. € †< ' *# #  "             @ † Contemporary and Emerging Topics. Austin/New York: Landes Bioscience/Springer Science Business Media; 2011:107-122.

10

MAST CELL BIOLOGY

• ` * *#      #  { @ ="   _•••– Œ• $ _—^Œ_  ` * \ ^   \  ‚   ‚   $<    % { smooth muscle function and remodeling. J Allergy Clin Immunol 2003; 111(1 Suppl):S18-34; discussion S34-16. _ ! =\ †  \    $ {          Cell Biology: Contemporary and Emerging Topics. Austin/New York: Landes Bioscience/Springer Science Business Media; 2011:235-269. 13. Kushnir K -Sukhov NM, Brown JM, Wu Y et al. Human mast cells are capable of serotonin synthesis and release. J Allergy Clin Immunol 2007; 119(2):498-499. 14. Stevens RL, Adachi R. Protease-proteoglycan complexes of mouse and human mast cells and importance  # ^ ^#  "  {   $ $ ‚! _••”– 217:155-167. 15. Schwartz LB. Tryptase: a clinical indicator of mast cell-dependent events. Allergy Proc 1994; 15(3):119-123. 16. Irani AM, Schwartz LB. Human mast cell heterogeneity. Allergy Proc 1994; 15(6):303-308. ” @$# `       !  {          DD, eds. Mast Cell Biology: Contemporary and Emerging Topics. Austin/New York: Landes Bioscience/ Springer Science Business Media; 2011:212-234. 18. Boyce JA. Mast cells and eicosanoid mediators: a system of reciprocal paracrine and autocrine regulation. Immunol Rev 2007; 217:168-185. 19. Boyce JA. Eicosanoid mediators of mast cells: receptors, regulation of synthesis and pathobiologic implications. Chem Immunol Allergy 2005; 87:59-79. 20. Kasperska-Zajac A, Brzoza Z, Rogala B. Platelet activating factor as a mediator and therapeutic approach   # # { _••—– Œ˜_™_^_• 21. Olivera A, Rivera J. An emerging role for the lipid mediator sphingosine-1-phosphate in mast cell effector $              @ † @    Emerging Topics. Austin/New York: Landes Bioscience/Springer Science Business Media; 2011:123-142. 22. Rivera J, Proia RL, Olivera A. The alliance of sphingosine-1-phosphate and its receptors in immunity. Nat Rev Immunol 2008; 8(10):753-763. 23. Zhang S, Anderson DF, Bradding P et al. Human mast cells express stem cell factor. J Pathol 1998; 186(1):59-66. 24. Okayama Y. Mast cell-derived cytokine expression induced via Fc receptors and Toll-like receptors. Chem Immunol Allergy 2005; 87:101-110. 25. Katsanos GS, Anogeianaki A, Orso C et al. Mast cells and chemokines. J Biol Regul Homeost Agents 2008; 22(3):145-151. 26. K Kulka M, Fukuishi N, Metcalfe DD. Human mast cells synthesize and release angiogenin, a member of the ribonuclease A (RNase R A) superfamily. J Leukoc Biol 2009; 86(5):1217-1226. 27. Freeman TA, Parvizi J, Dela Valle CJ et al. Mast cells and hypoxia drive tissue metaplasia and heterotopic    #  #      <  #    *$ ‚ Œ˜™” 28. Theoharides TC, Zhang B, Kempuraj D et al. IL-33 augments substance P-induced VEGF secretion from human mast cells and is increased in psoriatic skin. Proc Natl Acad Sci USA 107(9):4448-4453. 29. Stone KD, Prussin C, Metcalfe DD. IgE, mast cells, basophils and eosinophils. J Allergy Clin Immunol 125(2 Suppl 2):S73-S80. Œ• #$  @\  # ~ *#      ! $       DD, eds. Mast Cell Biology: Contemporary and Emerging Topics. Austin/New York: Landes Bioscience/ Springer Science Business Media; 2011:162-185. Œ ‚  @ ! =                Cell Biology: Contemporary and Emerging Topics. Austin/New York: Landes Bioscience/Springer Science Business Media; 2011:270-288. 32. Okayama Y, Kawakami T. Development, migration and survival of mast cells. Immunol Res 2006; 34(2):97-115. 33. K Kraft S, Kinet JP. New developments in FcepsilonRI regulation, function and inhibition. Nat Rev Immunol 2007; 7(5):365-378. 34. Kashiwakura J, Otani IM, Kawakami T. Monomeric IgE and mast cell development, survival and function.          @ † @    =  * $ New York: Landes Bioscience/Springer Science Business Media; 2011:29-46. 35. Kawakami T, Galli SJ. Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2002; 2(10):773-786. 36. Kawakami T, Kitaura J. Mast cell survival and activation by IgE in the absence of antigen: a consideration of the biologic mechanisms and relevance. J Immunol 2005; 175(7):4167-4173. Œ”     *<}< @    #%  ^ ! ~ ‚! $ _••“– 6(3):218-230.

MAST CELL BIOLOGY: INTRODUCTION AND OVERVIEW

11

Œ— $# `      ^$    #    ¡RI-mediated mast cell activation. Immunol Lett 2007; 113(2):59-69. 39. Tkaczyk C, Horejsi V, Iwaki S et al. NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc¡RI aggregation. Blood 2004; 104(1):207-214. ’• `$ *‚     *<}< @     ¡RI mediate unique and convergent signals for   {      #$   † _••’– •’˜—™_’•^_’” 41. Kuehn K HS, Radinger M, Brown JM et al. Btk-dependent Rac activation and actin rearrangement following Fc¡RI aggregation promotes enhanced chemotactic responses of mast cells. J Cell Sci 2010; 123(Pt 15):2576-85. 42. Qiao H, Andrade MV, Lisboa FA et al. Fc¡R1 and toll-like receptors mediate synergistic signals to markedly $  $  {  <  $    † _••“– •”˜_™“•^“— 43. Allakhverdi Z, Smith DE, Comeau MR R et al. Cutting edge: The ST2 ligand IL-33 potently activates and drives maturation of human mast cells. J Immunol 2007; 179(4):2051-2054. 44. Ho LH, Ohno T, Oboki K et al. IL-33 induces IL-13 production by mouse mast cells independently of IgE-Fc¡RI signals. J Leukoc Biol 2007; 82(6):1481-1490. ’   \! ‚     #      #" >   @ Immunol 2009; 124(4):639-646; quiz 647-638. ’“ & † ˆ! >  ƒ<  ^             eds. Mast Cell Biology: Contemporary and Emerging Topics. Austin/New York: Landes Bioscience/ Springer Science Business Media; 2011:91-106. ’” $ \ †  † `%<  ƒ  {${$ #    # !   membrane structure and function. Semin Cell Dev Biol 2007; 18(5):583-590. 48. Wilson BS, Steinberg SL, Liederman K et al. Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes. Mol Biol Cell 2004; 15(6):2580-2592. 49. Holowka D, Sheets ED, Baird B. Interactions between Fc¡RI and lipid raft components are regulated by the actin cytoskeleton. J Cell Sci 2000; 113 ( Pt 6):1009-1019. 50. Wilson BS, Pfeiffer JR, Oliver JM. Fc¡RI signaling observed from the inside of the mast cell membrane. Mol Immunol 2002; 38(16-18):1259-1268.  š# š @#  ~ $ $ š   @$   !  {$ ¡RI-induced mast cell activation and allergic responses. J Immunol 2007; 179(2):740-743. 52. Parravicini V, Gadina M, Kovarova M et al. Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat Immunol 2002; 3(8):741-748. 53. Choi WS, Hiragun T, Lee JH et al. Activation of RBL-2H3 mast cells is dependent on tyrosine phosphorylation of phospholipase D2 by Fyn and Fgr. Mol Cell Biol 2004; 24(16):6980-6992. 54. Hong H, Kitaura J, Xiao W et al. The Src family kinase Hck regulates mast cell activation by suppressing an inhibitory Src family kinase Lyn. Blood 2007; 110(7):2511-2519. 55. Silverman MA, Shoag J, Wu J et al. Disruption of SLP-76 interaction with Gads inhibits dynamic clustering of SLP-76 and FcepsilonRI signaling in mast cells. Mol Cell Biol 2006; 26(5):1826-1838. 56. Gu H, Saito K, Klaman LD et al. Essential role for Gab2 in the allergic response. Nature 2001; 412(6843):186-190. 57. Yu M, Lowell CA, Neel BG et al. Scaffolding adapter Grb2-associated binder 2 requires Syk to transmit signals from Fc¡RI. J Immunol 2006; 176(4):2421-2429. 58. Iwaki S, Spicka J, Tkaczyk C et al. Kit- and Fc¡RI-induced differential phosphorylation of the transmembrane  $ ~*ƒƒ†ƒ*_ % {"    $    @ Signal 2008; 20(1):195-205. 59. Linnekin D. Early signaling pathways activated by c-Kit in hematopoietic cells. Int J Biochem Cell Biol 1999; 31(10):1053-1074. 60. Ozawa K, Yamada K, Kazanietz MG et al. Different isozymes of protein kinase C mediate feedback inhibition of phospholipase C and stimulatory signals for exocytosis in rat RBL-2H3 cells. J Biol Chem 1993; 268(4):2280-2283. 61. Furuichi T, Yoshikawa S, Miyawaki A et al. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature 1989; 342(6245):32-38. 62. Ma HT, Beaven MA. Regulators of Ca2 signaling in mast cells: potential targets for treatment of mast-cell  ›          @ † @    =  Topics. Austin/New York: Landes Bioscience/Springer Science Business Media; 2011:62-90. 63. Liou J, Kim ML, Heo WD et al. STIM is a Ca2 sensor essential for Ca2 -store-depletion-triggered Ca2

{$" @$ † _••– ˜Œ™_Œ^_’ 64. Roos J, DiGregorio PJ, Yeromin AV et al. STIM1, an essential and conserved component of store-operated Ca2 channel function. J Cell Biol 2005; 169(3):435-445.

12

MAST CELL BIOLOGY

“ <  %< š \ <     $  ˆ  $ $      @‚@ channel function. Nature 2006; 441(7090):179-185. 66. Vig M, Peinelt C, Beck A et al. CRACM1 is a plasma membrane protein essential for store-operated Ca2

entry. Science 2006; 312(5777):1220-1223. 67. Ma HT, Beaven MA. Regulation of Ca2 signaling with particular focus on mast cells. Crit Rev Immunol 2009; 29(2):155-186. “—   ‚      *# $   ## Œ^<     Trends Immunol 2008; 29(10):493-501. 69. Ali K, Bilancio A, Thomas M et al. Essential role for the p110b phosphoinositide 3-kinase in the allergic response. Nature 2004; 431(7011):1007-1011. 70. Laffargue M, Calvez R, Finan P et al. Phosphoinositide 3-kinase a         function. Immunity 2002; 16(3):441-451. 71. Tkaczyk C, Beaven MA, Brachman SM et al. The phospholipase C a1-dependent pathway of Fc¡RI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase. J Biol Chem 2003; 278(48):48474-48484. ”_ %<  *<}< @  #% †   †<    $   #    ¡RI-mediated mast cell activation by kit. J Biol Chem 2005; 280(48):40261-40270. 73. Karra L, Levi-Schaffer F. Down-regulation of mast cell responses through ITIM containing inhibitory            @ † @    =  * Austin/New York: Landes Bioscience/Springer Science Business Media; 2011:143-161. 74. Karra L, Berent-Maoz B, Ben-Zimra M et al. Are we ready to downregulate mast cells? Curr Opin Immunol 2009; 21(6):708-714.

SECTION II

MAST CELL DEVELOPMENT AND HOMEOSTASIS

CHAPTER R2 MAST CELL PROGENITOR TRAFF R ICKING AND MATURA R TION Jenny Hallgren1  #  $ #|,2 1

Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden; Harvard Medical School and Brigham and Women’s Hospital, Boston, Massachusettes, USA          2

Abstract:

Mast cells are derived from the hematopoietic progenitors found in bone marrow and spleen. Committed mast cell progenitors are rare in bone marrow suggesting they are rapidly released into the blood where they circulate and move out into the  #  $ *#        $     †   <  #  ‹$  "   _4`7 integrin and the chemokine receptor CXCR2 by the mast cell progenitors and expression of MAdCAM-1 and VCAM-1 in the intestinal endothelium; and is also controlled by dendritic cells expressing the transcriptional regulatory protein T-bet. None of these play a role in    <  # $ &# # $    {  # $ there is marked recruitment of committed mast cell progenitors to lung and these cells must express _4`7 and _4`1 integrins. Within the lung there is a requirement for expression of VCAM-1 on the endothelium that is regulated by CXCR2, also expressed on the endothelium. There is a further requirement for expression of the CCR2/CCL2 pathways for full recruitment of the mast cell progenitors to the ^{ $

INTRODUCTION Mature mast cells are found in many connective tissues where they are rare long-lived cells. These constitutive and connective tissue-associated mast cells have a restricted phenotype based on mediator production and secretory granule proteases that varies somewhat from tissue to tissue. An entirely different phenotype, prominent after a   %     # $    %# #  mucosa. This rodent mucosal phenotype also shows some variability in granule content.

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 14

MAST CELL PROGENITOR TRAFFICKING AND MATURATION

15

~# #    $ $    # #    %    cells, the connective tissue and the mucosal phenotypes. In general, two broad classes     #!     # #$       in secretory granule protease expression. All phenotypes appear to be derived from a      %# #  #     ^!  factors such as cytokines or perhaps by interactions with the extracellular matrix.1-3 In this chapter we address the issues of how mast cells arise and how they get to their diverse sites of residency. Most of the discussion will be focused on studies conducted in the mouse but wherever possible, differences and similarities in the human will be mentioned.

MAST CELL DEVELOPMENT The seminal studies by Kitamura and colleagues in the late 1970s showing $      ^    # !    % type bone marrow indicated these cells could be derived from the bone marrow and thus were of hematopoietic origin.4,5 Then, in the early 1980s, the ability to grow cultured mast cells from murine bone marrow using Interleukin (IL)-3 was discovered.6,7 This propelled the studies on understanding the developmental pathway and the observation that the immature mast cell progenitors could be found in peripheral tissues such as in the intestine.8-10 In the last few years, a number of studies by Akashi and colleagues as %  #  #!   !   #     %# #  been termed the classical model of hematopoiesis.11-14 *#   # ! of the mast cell in mouse bone marrow occurring along the myeloid pathway. In this model, the initial fate decision made by the hematopoietic stem cell is to commit to the   #  *#     !  #  } by the down regulation of the Sca-1 antigen. This intermediate has been termed the Common Myeloid Progenitor (CMP) and is distinguished from the Common Lymphoid Progenitor by expression of IL-7R R by the latter cells. The CMP intermediate can give rise to either the Megakaryocyte-Erythrocyte Progenitor or to the next step in myeloid development, the Granulocyte Macrophage Progenitor (GMP) which is distinguished by # $ $  "   # %      a‚ ˜  by the 2.4G2 mAb). The GMP can give rise to macrophages, eosinophils, neutrophils  # ! †#^  @ \  ˜† @\™     #   C57BL/6 mice. These BMCP, were only found in the spleen of this mouse strain and $      * , FcaRII/III , `7 integrinhi, Fc¡RI< cells, that only gave rise to mast cells or basophils in culture despite using cytokines that give rise to all the myeloid cells after culture of bone marrow cells. Furthermore, transfer of these BMCP into mast ^     #       #     ! demonstrating their capacity was not limited to in vitro differentiation.12 The isolation of the CMP, GMP and BMCP; and the ability of these cells to differentiate into basophils  $$  #!     # 15 A second mast cell differentiation pathway in adult mice has also been described in which the cells are derived from the multipotential progenitor.16 In these experiments, the authors isolated the mast cell progenitors as lineage negative (including lacking the ##   =   ¡RI), CD27–, `7 integrin , IL33R R (T1/ST2) . This fraction, as with the BMCP, was able to restore mast cells but not other lineages after transfer into  ^    #   %   †  #

16

MAST CELL BIOLOGY

 # $#            #    occurring via a direct commitment from the multipotential progenitor. This development scheme is more in keeping with the alternative model of hematopoietic development suggested by Kawamoto and others.17-19 *#  %  ! $  liver-derived cells but has been extended to adult hematopoiesis as well. It suggests that  !     #      in this model, myeloid potential is retained even past the differentiation between B and T-lymphocyte commitment. Support for this comes from many investigations both  !    !!    $#  #   # $  Œ•œ  # macrophages in the thymus are derived from the early T cell progenitors migrating into the thymus from the bone marrow.17 In either scheme, it is thought the committed mast cell progenitor is released into the blood stream where it migrates to various tissues as discussed further below. While there has been little investigation of the blood-borne mast cell progenitor in adult mice,  €€“ ‚%      #         in fetal mouse blood.20 *#     * , Thy-1lo was unusually prevalent     %    *# $  $‹$   % suggesting the cells were perhaps seeding tissues at a particular stage in development. ! #  $        $    $    that there is an important developmental period where mast cells seed tissue such as the skin and provide the indigenous cell population with little turn over.21 The surprising ease with which murine bone marrow gave rise to mast cell lines in vitro was not initially reproducible with human hematopoietic progenitors. It awaited our understanding of the importance of stem cell factor (SCF, also called KIT ligand, KITl) in the growth and differentiation of mast cells before routine in vitro culture of mast cells from human cord blood was established.22-24 This is now an established technique for studying human mast cells in vitro and has been used in many studies such as to demonstrate # # #$     #       #     related to macrophages in development and is increased in the blood of asthmatics.25-27 *#     @    ! %  $  # !  of mutations in the kit $     # W locus, which resulted in a mast cell    !     #   $ *    $    $    # $ $         of mast cells in the peripheral tissues. The ability to generate early myeloid progenitors is not eliminated, however, leaving the ability to develop mast cells in culture via IL-3 intact.28 ~# # {$  @  !     ! $  of the cells toward a connective tissue phenotype and sustaining the cells both in vitro and in vivo.29-31 Unfortunately, as this receptor is involved in myelopoiesis, gametogenesis and    #  $    #       Among other affects, there are decreased numbers of basophils in the bone marrow and † @\  # –    $   $  %##  {$ $  on responses involving these two cells,12,32 Thus, caution is necessary in interpreting the $  $ *^  @^     ! ‚%    !   #  #    a committed mast cell progenitor. This Thy-1lo, KIT fetal blood cell responded only to SCF and IL-3 together in vitro and lacked any potential for becoming macrophages, granulocytes, erythrocytes or B or T-lymphocytes in several different assays, but gave       ##  ‹$ *   #     

MAST CELL PROGENITOR TRAFFICKING AND MATURATION

17

cell progenitors in adult mouse (or human) blood has not been completed, although we #!          # # $   $ as lineage-, CD45 , CD34 , Fc¡RI , `7 integrinhi cells and a c-kit , CD34 macrophage/ mast cell progenitor is found in human peripheral blood.12,25,33 Of note is the continued expression of the CD34 marker of murine mast cells throughout their maturation, although this is usually a marker of early hematopoietic progenitors.34,35

BASAL HOMING OF MAST CELL PROGENITORS

 $       < #! $  !  #  intestine or peritoneal cavity. This is because of the relatively large number of committed mast cell progenitors or easily obtainable mature mast cells, respectively, found at these peripheral tissue sites in rodents. The pool of committed mast cell progenitors in the intestine is greater than that found in bone marrow (among mononuclear cells isolated from the tissues), although there are many more mononuclear cells and therefore cells within this lineage in the bone marrow. This large intestinal pool of committed mast cell progenitors facilitated the initial studies identifying these cells using clonogenic assays with IL-3 alone or in combination with SCF.8,10,36-38 It also facilitated our investigations on the molecules critical to establishing and maintaining this pool. The appreciation of the immature mast cell progenitor was a direct consequence of the early studies highlighting the ability of these cells to be expanded and maintained with IL-3. This led  $  !   #% #           lung and intestine.8,36,39 While the early studies used a clonogenic assay that did not distinguish early precursors from the committed mast cell progenitors, the assay did % #    ^   !$   # $ that a population of committed mast cell progenitors is found in bone marrow, spleen, lung and intestine.12,16,33 *#    `7 integrins on rodent and human mast cells suggested a role for one or both of the `7 integrins, _4`7 or _E`7 in homing of mast cells to the intestine based on their involvement in the homing of lymphocytes to the small intestine and associated lymphatic structures.40-43 To test this hypothesis, we used both null strains and blocking antibodies directed against the various integrin chains. These techniques further allowed $    #     #   #   #<  and a role for dendritic cells in the establishing and maintaining of the large intestinal pool of committed mast cell progenitors. Using the `”   $ % $   $   #        #%  #   %     # $    #  37,44 The    $    %  # $   # $$ ! tissue and in the mucosa of the intestine. This lack of both phenotypes in the absence of progenitors supported the paradigm that a single committed progenitor gives rise to all phenotypes of mature mast cells. In order to address the issue of which of the `7 integrin molecules was critical in controlling ingress, we established a protocol to allow the use of antibody blocking. To do this as, we took advantage of the observation made a number of years earlier by Kitamura and colleagues, that the progenitors are very sensitive to irradiation.45 This allowed us to eliminate the intestinal pool of mast cell progenitors and # $  $ ! # $   # " _ %< & $ {$ # mast cell progenitor reconstitution via the administration of antibodies to the different

18

MAST CELL BIOLOGY

integrins. Using anti-_4, anti-`1, anti-`7, anti-_E and anti-_4`7 integrin antibodies, # !   %    _4`7 and not _4`1 or _E`7. Although after entry, the intestinal-localized mast cell progenitors down regulate expression of _4`7 and up regulate expression _E`7.46 As the _4`7 integrin molecule can bind to both the mucosal addressin cellular adhesion molecule (MAdCAM)-1 or vascular cell adhesion molecule (VCAM)-1, both of which are expressed within the intestine, we evaluated the involvement of each cellular adhesion molecule. Using both null mice lacking VCAM-1 or antibody blocking directed to MAdCAM-1 or VCAM-1, both molecules were showed to be required for the establishment of the intestinal pool of mast cell progenitors. We also evaluated various #<  $  <    #< #% !!  #   & $   !     @@‚_ @@‚Œ  @@‚ %#   # %  !  @@‚_ $ 38 This IL-8 receptor is expressed on both circulating leukocytes and on endothelium, suggesting it could be affecting the   < # # $#   #  $       # !$ endothelium.47 Adapting the techniques of sublethal irradiation followed by bone marrow reconstitution of wild type mice that we used for antibody blocking, we reconstituted # ! $  %# # %   @@‚_^    % *# $ demonstrated reduced reconstitution in the wild type mice given CXCR2 null bone marrow, indicating that the circulating progenitor needs to express CXCR2. Further investigation also noted a role for CXCR2 expression by the host, as reconstitution of CXCR2 null mice with wild type bone marrow also resulted in fewer intestinal mast cell progenitors when compared to wild type mice reconstituted with wild type bone marrow in parallel.46 *#    $ }  $   The process of reconstitution of the pool of mast cell progenitors in the intestine occurred fairly rapidly after sublethal irradiation and reconstitution with wild type bone marrow, achieving about 50% of the normal frequency by one week and almost complete reconstitution by two weeks.37 This rapid replenishment of the pool suggested that this $           @   # ## came with the treatment of normal BALB/c mice with an antibody directed to either the _4`7 integrin or to the CXCR2 receptor. In both cases, administration of the antibodies every other day for about one week resulted in a ¾75% depletion of the pool of MCp.38 Since the cells down regulated _4`7 integrin expression and up regulated _E`7 integrin expression after entering the intestine, the depletion of the pool was a consequence of inhibition of the transmigrating cells attempting to leave the circulation rather that a direct effect on progenitors in the intestines. Support for this interpretation was obtained by monitoring the pool of mast cell progenitors in the lung. This pool is not dependent on either _4 integrins or expression of the CXCR2 receptor; and the size of this pool as well as that in spleen increased when mice were given anti-_4 integrin, suggesting an increase in number of circulating mast cell progenitors as a consequence of preventing them from moving into the intestine. Furthermore, the remarkable depletion of almost 80% after 6 days of treatment with the blocking monoclonal antibody of the pool suggested a halff life for the cells in the intestine of about 4 days. *#  $   #  #        #  %  suggested by Guy-Grand et al in their studies of this process.10 Using germ free mice, they found there was no diminution in the size of progenitor pool in the absence of intestinal {  $ #   # }    % $   #  %## < mature T cells. We pursued this further using mice lacking the recombinase activating

MAST CELL PROGENITOR TRAFFICKING AND MATURATION

19

Figure 1. Development of committed mast cell progenitors (MCp) in the bone marrow (BM) from the #   ˜`@™  # $        #   <  # #   #     ! $  ^{  *%   developmental pathways have been proposed leading to the production of committed mast cell progenitors. *#  # ! $   #  $ ! #  $     #   $  $#  #     ! $ #      $ $#  # _4 integrins and various chemokine pathways. A role for the C3b and TPO receptors has been shown to affect mast cell numbers in the skin and peritoneal cavity. See text for details.

gene (RAG)-2 and the common IL-R R gamma chain (common for IL-2, IL-4, IL-7, IL-9  ƒ^  ™ *# $    <  $  # $ natural killer, T, B and natural killer T cells.48 As in athymic mice, mast cell progenitors homed to the intestine in normal numbers in the absence of all of these other cells types. *# %        #  # @”†ƒ“  †ƒ†  had normal numbers of intestinal mast cell progenitors in the absence of T cells.37 Given this lack of dependence on the adaptive immune system, we had assumed this was an       #   *#$  % $     # in mice lacking the T helper Type 1 master developmental regulator, the transcription factor T-bet (Tbx21), there were decreased numbers of mast cell progenitors in both the lung and intestine of both C57BL/6 and BALB/c mice. This also resulted in fewer numbers of mature intestinal mast cells as expected from previous studies.49 These results indicated an innate cell type must be involved and affected developmentally by the loss of T-bet. Yet there was no evidence of expression within the mast cell lineage, despite  # *^ $   #%   #  ž@ ^ $ {% *# demonstration that dendritic cells also expressed T-bet and that loss of this protein had an effect on their role in an arthritis model led us to consider whether this cell type played some role in the formation of the intestinal pool of mast cell progenitors.50 Unexpectedly, the transfer of bone marrow-derived dendritic cells from wild type mice to wild type or

20

MAST CELL BIOLOGY

T-bet null mice restored the pool of intestinal mast cell progenitors to similar levels in #   *#$ %# #      #  #   <    progenitors to the lung or intestine, dendritic cell functions or development controlled by *^       #   <  #       #  and development of mature mast cells in this tissue. In subsequent work, we have repeated these experiments, evaluating the much smaller pool of mast cell progenitors in the lung and found no reconstitution of the pulmonary pool in T-bet null mice by wild type bone marrow-derived dendritic cells (Moravia et al, unpublished observations). Thus, while these studies established a critical role for T-bet dendritic cells in controlling the numbers of mast cells in the intestine, this is not the case for the lung. It is remarkable that, as of this time, very little is known about what controls the basal pool of mast cell progenitors in the lung. Other studies have also implicated a role for CD11b and the TPO receptor Mpl on maturation and movement of the mast cell progenitors into the peritoneal cavity and skin. Thus, reduced numbers of mast cells were found in the peritoneal cavity and dorsal <  @^  51 However, there was no defect in the number of mast cell progenitors noted in bone marrow or intestine, suggesting that the loss of this C3b receptor plays a role in maturation or survival of the cells at these sites and does not {$   <  #     $ #  ˜*\ˆ™ #  shown to increase human mast cell colonies in vitro and increase apoptosis of murine mast cells both in vitro and in vivo, TPO receptor (Mpl) null mice were evaluated for number of mast cell progenitors and mature mast cell in various sites. Using the Chen et al      %       ¡RI–, CD27–, `7 integrin and IL33R , loss of Mpl resulted in decreased numbers of mast cell progenitors in the spleen and bone marrow, but increased numbers of mature mast cells based on histological   {$    *  ¡RI.16,52,53 There were also increased numbers of mast cells in the spleen, bone marrow, skin, stomach and peritoneal cavity, suggesting that TPO via its receptor plays an important role in controlling the number of mast cell in several different sites.

RECRUITMENT OF MAST CELL PROGENITORS

  $       $ $  {   such as in the joints with arthritis or in the lungs of patients with allergic asthma.54-56 In asthmatics, this is accompanied by an increase in circulating progenitor cells as assessed in a clonogenic assay.27 In mice, the normal number of mast cells in the airways of the common wild type strains C57BL/6 and BALB/c is relatively low, $       # $     % {57,58 This increase may be due to either division of the few mature mast cells in situ or recruitment and maturation of mast cell progenitors in the lung, or both. Because of # $!   < $    #      previous studies suggesting recruitment to the intestine after helminth infection in mice,10,59 % ##} # #      {  $  mast cell progenitors via transendothelial migration from the blood to the lung. We " #    !   #  $ $  # {   # %# # $   !!   #% $#  $     <  #  *  # # †ƒ†  @”†ƒ“  % 

MAST CELL PROGENITOR TRAFFICKING AND MATURATION

21

immunized systemically with ovalbumin (OVA) adsorbed to alum on day 0 and day 7, followed by three consecutive daily OVA aerosol challenges starting 10 days later. Both   #!           # $  # %# a magnitude of ¾ 28 fold increase in total number of pulmonary mast cell progenitors in BALB/c mice and ¾ 10 fold increase in C57BL/6 mice.60 As reported by others, the #   {   #  ˆž     $     { %# @”†ƒ”“  #%    { #  †ƒ†61 The magnitude of the increase in such a short period of time after the initial antigen challenge indicted that recruitment rather than expansion of the small numbers of resident progenitors was the most likely explanation    # !    $   #   #   $  ˆž^$ $ { ž@ ^ "   # $ endothelium is up regulated.62 Using endothelial VCAM-1 knock out mice and anti-VCAM-1 treatment before each OVA-aerosol challenge, we showed that mast cell progenitor recruitment is critically dependent on VCAM-1 expression on the endothelium in the lung. In contrast to the homing of mast cell progenitors to intestine, there was no role for MAdCAM-1 in the recruitment to lung, consistent with reports that MAdCAM-1 is not expressed in the lung endothelium.63,64 As both _4`1 and _4`7 integrins can mediate binding to VCAM-1, we investigated the effect of blocking antibodies to these targets administered during the challenge phase. We found using several different antibodies that either integrin could reduce the mast cell progenitor recruitment, suggesting that both these integrins contribute in the adhesion to VCAM-1. Administration of antibody to _E, the other alpha chain that pairs with `7, had no effect,  #    # <  !   }  # `”^      # $  $      $ compared to their wild type controls. We concluded that mast cell progenitors express functional _4`1 and a4`7 integrins that interact with VCAM-1 on the lung endothelium to accomplish the transendothelial migration (Fig. 2).

Figure 2.    # $     ˜  #    ƒ*†4, PGE2) {^$  $  @  # $  †ƒ†   {$ # $   $  $   Ÿ$  <  #%     #  !! See text for details.

22

MAST CELL BIOLOGY

As recruitment also requires attracting the mast cell progenitor to the site of the { %   # ^$      $  several different knock out mouse strains lacking the chemokine receptors CXCR2, CCR5, CCR3 and CCR2. We focused on these based on their known expression by bone marrow derived mast cells (BMMC) and/or human cord blood derived mast cells.24,65,66 Using our established mast cell progenitor recruitment protocol, we found no diminution in mast cell progenitor recruitment in CCR5 and CCR3 knockout mice. CXCR2–/–  #%!  #   $      $ 66% in total mast cell progenitor content and 53% in the concentration of mast cell progenitors per 106 mononuclear cells.33 *   # #   @@‚_ % $ to lack of expression on the mast cell progenitor, we made bone marrow chimeras of wild type mice reconstituted with CXCR2–/– bone marrow. To our surprise, the CXCR2–/– bone marrow could reconstitute the mast cell progenitor recruitment in sublethally irradiated wild type mice, indicating that CXCR2 expression by the circulating mast cell progenitor was not needed. The opposite experiment where bone marrow chimeras of CXCR2–/– mice were produced by being given wild type bone marrow recapitulated the original results with the CXCR2 knockout mice indicating that a nonbone marrow-derived, relatively radioresistent host cell had to express CXCR2 for recruitment of mast cell progenitor to lung. Because CXCR2 is expressed on the lung endothelium, we hypothesized that CXCR2 expression on the lung endothelium regulates VCAM-1 expression. In accordance with this hypothesis, total mRN R A levels in the lungs and protein expression of VCAM-1 on the lung endothelium was strongly $  @@‚_^   $ # @@‚_  # $ #$  $ ž@ ^ "   #  # $ {$"       to this tissue. In support of our observations, endothelial CXCR2 was also shown to,    $ $ # {$"  ƒ\^$ $ ‘$ 47 Evaluation of CCR2 null mice also noted reduced recruitment in our model and consistent with this, we found that the chemokine CCL2, the major ligand for CCR2 is chemotactic for bone marrow-derived mast cell progenitors and immature (2 weeks old) BMMC cultured in SCF and IL-3. However, CCL2 was not chemotactic to BMMC cultured in IL-3 alone, even though mast cells cultured in both conditions expressed @@‚_  $  @@ƒ_ %# #   67 This indicates that SCF is an important mediator in coupling CCR2 to downstream signaling events that leads to chemotaxis. When CCL2 was injected intradermally into mice, immature BMMC from IL-3 SCF cultures administered intravenously migrated to the sites of injection. These data suggested that the CCL2/CCR2 pathway was important in recruitment of mast cell progenitors; and this was supported when we demonstrated that both CCR2 @@ƒ_^   #   $       $  # $  $      % { `%!  %# %   whether this was due to expression of CCR2 by the mast cell progenitors by evaluating recruitment in sublethally irradiated wild type mice given CCR2–/–– bone marrow, we found normal recruitment, showing that CCR2 expression by the circulating progenitors   & $ #   #  } !   #    in the reconstituted wild type mice and found them to all be of donor (CCR2–/–) origin. The opposite experiment, where sublethally irradiated CCR2–/–      ! wild type bone marrow, still had diminished recruitment, indicating a requirement for expression of the receptor by host tissues. Since mice lacking CCL2, also had diminished mast cell progenitor recruitment to lung, we also evaluated these mice using bone marrow

MAST CELL PROGENITOR TRAFFICKING AND MATURATION

23

chimeras. We found that the critical source of CCL2 was both in bone marrow-derived cells and stroma-derived cells. Clearly, the CCL2/CCR2 axis is involved in the mechanisms that lead to antigen-induced mast cell progenitor recruitment to lung, but the process is "  #          $  !      the critical chemokine receptor(s) and the mechanism(s) by which they act to attract the mast cell progenitor to lung in vivo, the hunt is not over. Several other chemoattractants for mast cells may also be involved. In a study    # #         $      ˆž^$  % { †ƒ†  %  } %< %# #   ‘ %# ˆž  $ %  !    #      # #    %# ˆž &  $ <  $ _• #   ˆž #     $ conditional medium that was used to attract immature (2-week-old) BMMC produced in IL-3 alone.68 \$    # #  !  # $  medium demonstrated that prostaglandin E2 (PGE2) was inducing the chemotactic response and that an EP3 receptor antagonist could block the response. In addition, LTB4 was shown to be chemotactically active on immature (2-week-old) BMMC.69 Because both mediators are expressed following activation of mast cells, they are most likely to play a role in the recruitment process rather than the basal homing. Furthermore, the demonstration of an effect on 2-week-old cultured mast cells by CCL2, LTB4 and PGE2 with only PGE2 attracting the more mature 10-week-old cultured mast cells, suggests a role for these factors in early recruitment, perhaps from the bone marrow rather than into the tissues. Mast cell progenitor recruitment to allergen-challenged lung occurs in an ^     !$ }   # $  $  the sensitization process skews the immune response towards a Th2 response, we hypothesized that Th2 cells control mast cell progenitor recruitment by providing Th2 < # !  # {^$ $^ $  # $ and/or necessary production of chemoattractants. In support of this we found that the pulmonary mast cell progenitor recruitment was dependent on CD4 T cells in BALB/c mice, since depletion with monoclonal antibody before aerosol challenge inhibited the recruitment but depletion of CD8 T cells had no effect.70 Unexpectedly, BALB/c mice lacking IL-4, IL-4R R alpha chain or STAT-6 had a normal recruitment response, even though these mouse strains have diminished Th2-responses and no serum IgE.71,72 Further, administration of blocking antibodies for the Th2 cytokines IL-3, IL-4, IL-5, IL-10 or IL-13 during the challenge phase had no effect on the recruitment. These data suggested that a Th2-response is not required for the antigen-induced recruitment of mast cell progenitors to the lung. Similarly IFN-a–/–, IL-12p40–/–, IL12R`1–/–– mice or treatment with anti-IFN-a, anti-IL-12p40 (the IL-12 and IL-23 common chain), anti-IL-6, anti-IL-17A and anti-TGF`1 did not reduce the recruitment, indicating no role for Th1 or for Th17 cells. Furthermore, anti-CD25 treatment had no effect on the mast cell progenitor recruitment, ruling out a critical role for T-regulatory cells. As natural killer T (NKT) cells have been implicated in the allergic pulmonary allergic response, we next evaluated a role for these cells.73,74      $  @^   %## <  ~*  #   $ $   $   cell progenitors. Monoclonal antibody blocking of CD1d during the challenge phase    $ #      $    %  mice treated with isotype-matched control immunoglobulins. Blockade or genetic loss

24

MAST CELL BIOLOGY

of CD1d signaling prevents the activation of both the Type 1/invariant NKT (iNKT) and the Type 2 or diverse NKT cells. However, J_—^   #   < iNKT cells can be used to differentiate between these two types of CD1d-restricted NKT cells. Surprisingly, we found that the mast cell progenitor recruitment was greater in the antigen-sensitized and challenged J_—^   #  # %    but that anti-CD1d blocking antibody still reduced the mast cell progenitor numbers in this mutant mouse strain, indicating that it is the Type 2 NKT cells that is critical for the mast cell progenitor recruitment to the antigen challenged lung. The screening of possible critical cytokines produced by CD4 T cells eliminated many of the mast cell-activation factors we thought might be involved. This led us to IL-9, a mast cell growth factor that is not dependent on STAT-6 for signaling.75 Use of ƒ^€     %  ^ƒ^€   %#   $  # # #   $      $ ˆ  source of pulmonary IL-9 is tissue resident mature mast cells. Using three different mast ^  $   ˜&sh/Wsh, W/Wv and Sl/Sld), we found that all strains had increases in mast cell progenitor recruitment comparable to their wild type controls, thereby excluding mature mast cells as the critical source of IL-9. Since NKT cells from C57BL/6 mice also had been suggested as a source of IL-9,76 % $   ~* cells using alpha-Galactosylceramide-CD1d tetramers, from BALB/c mice, cultured them with alpha-GalCer-pulsed bone marrow-derived dendritic cells and evaluated the supernatants for production of IL-9. These cells produced nanogram amounts of IL-9 in vitro, comparable to that produced by cultured polarized Th2 cells. To address if the requirement for IL-9 and for NKT cells were in the same pathway or parallel, we treated ƒ^€^   %   %# ^@ <   @^  and wild type mice with anti-IL-9 blocking antibody. No further reduction of the partial suppression of mast cell progenitor recruitment occurred by combining the inhibition of these two factors. These data indicates that IL-9 and the Type 2 CD1d-restricted NKT cells function in the same pathway.70

MAST CELL HYPERPLASIA The increased numbers of mature mast cells associated with parasitic nematode infections and chronic allergic reactions is likely the direct result, at least in part, of the maturation of the increased numbers of mast cell progenitors called into these   { &# $  $   "  #       # contribution of resident versus recruited mast cells, several studies have contributed  $ <%    # @@‚_ $  #   {$"    progenitors observed after only 3 aerosolized allergen challenges was associated with fewer numbers of interepithelial mast cells observed in the tracheal mucosa a week after the last challenge.33    #  %       # $  $$ $!   *#  $     %## # recruited mast cell progenitors give rise to the mature mucosal mast cells, but not the connective mast cell. Further work is necessary to establish the relative expansion of the connective tissue mast cells versus the relative contribution of the recruited progenitors to the increased number of mature cells. Other factors that have been implicated in the formation of the mast cell hyperplasia include the T cell derived factors IL-3, IL-4, IL-9 and more recently IgE as a survival

MAST CELL PROGENITOR TRAFFICKING AND MATURATION

25

and anti-apoptotic factor.77-79 Using antibody blocking and null mice, IL-3, IL-4 and IL-9 all have been show to be required for the mucosal mast cell hyperplasia associated with a nematode infection.77,78,80 $#  #   #$#  # {$  mucosal mast cells in the lung. The critical role for IL-9 in the recruitment of the mast cell progenitor to the lung was discussed above and these studies also eliminated a role for IL-3 and IL-4 in this process in BALB/c mice. Further studies have suggested that  ƒ^€^   #  $ $       # $  of mature mucosal mast cells, which may in turn suggest that the early dependence of recruitment is lost with further stimulation. Because of the role for IL-3 and IL-4 in the mature mast cell hyperplasia in the intestine, these bear further investigation in the lung. Recently, a role for IgE has been found in the mast cell maturation process occurring $  { '    $  { $  Aspergillus fumigatus extract that leads to remodeling and airway hyperresponsiveness, we evaluated the increase in both mast cell progenitors and the mature mast cells. Surprisingly, in BALB/c mice lacking IgE, recruitment of the immature progenitors was unaltered, but # $  $  $    %    79 The lack of IgE was further shown to lead to increased numbers of apoptotic cells, indicating that it acts as survival/anti-apoptotic factor.

CONCLUSION AND FUTURE PROSPECTS During homeostatic conditions, mast cell progenitors arise in the bone marrow and are transported out to peripheral tissues via the blood. It is likely that these circulating progenitors have already made the commitment to the mast cell lineage in the adult animal, $ # #      =" !   #    $  $      $     %# { *#  # to the intestine is highly controlled by other innate cells and by several different integrins, cellular adhesion molecules, chemokines and chemokine receptors. Many of these same    !!    # {^$  $  # $ $   # #%    $ $    # {^$ recruitment is dependent on other unique cells and factors that control the recruitment of #      $           $  that these mast cell progenitors mature into mast cells in situ and provide the basis for #   #   %#   {  $ `%!  # process is in need of much further work to understand the underlying mechanisms to be able to modulate this process and control the pathobiologic consequences of chronic activation of mast cells.

ACKNOWLEDGEMENTS We would like to thank the many collaborators who have participated in and supported this work. This work has been supported by grants from the National Institutes of Health to MFG (R01- AI083516).

26

MAST CELL BIOLOGY

REFERENCES 1. Kitamura Y, Yokoyama M, Matsuda H et al. Spleen colony-forming cell as common precursor for tissue mast cells and granulocytes. Nature 1981; 291(5811):159-160. 2. Gurish MF, Pear WS, Stevens RL et al. Tissue-regulated differentiation and maturation of a v-abl-immortalized mast cell-committed progenitor. Immunity 1995; 3(2):175-186. 3. Metcalfe DD, Baram D, Mekori YA. Mast cells Physiol Rev 1997; 77(4):1033-1079. 4. Kitamura Y, Shimada M, Hatanaka K et al. Development of mast cells from grafted bone marrow cells in irradiated mice. Nature 1977; 268(5619):442-443. 5. Kitamura Y, Go S, Hatanaka K. Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood 1978; 52(2):447-452. 6. Nagao K, Yokoro K, Aaronson SA. Continuous lines of basophil/mast cells derived from normal mouse bone marrow. Science 1981; 212(4492):333-335. 7. Razin E, Cordon-Cardo C, Good RA. Growth of a pure population of mouse mast cells in vitro with conditioned medium derived from concanavalin A-stimulated splenocytes. Proc Natl Acad Sci USA 1981; 78(4):2559-2561. 8. Crapper RM, Schrader JW. Frequency of mast cell precursors in normal tissues determined by an in vitro assay: antigen induces parallel increases in the frequency of P cell precursors and mast cells. J Immunol 1983; 131(2):923-928. 9. Schrader JW, Scollay R, Battye F. Intramucosal lymphocytes of the gut: Lyt-2 and thy-1 phenotype of the granulated cells and evidence for the presence of both T-cells and mast cell precursors. J Immunol 1983; 130(2):558-564. • $^     ƒ$$   $ $   ˆ        > =" Med 1984; 160(1):12-28. 11. Akashi K, Traver D, Miyamoto T et al. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 2000; 404(6774):193-197. 12. Arinobu Y, Iwasaki H, Gurish MF et al. Developmental checkpoints of the basophil/mast cell lineages in adult murine hematopoiesis. Proc Natl Acad Sci USA 2005; 102(50):18105-18110. Œ %< ` }$    ‚      # ^     # murine bone marrow. J Exp Med 2005; 201(12):1891-1897. ’   & ƒ <#       #     $ bone marrow. Cell1997; 91(5):661-672. 15. Ohmori K, Luo Y, Jia Y et al. IL-3 induces basophil expansion in vivo by directing granulocyte-monocyte progenitors to differentiate into basophil lineage-restricted progenitors in the bone marrow and by increasing the number of basophil/mast cell progenitors in the spleen. J Immunol 2009; 182(5):2835-2841. “ @# @@   *            $  \  ~ Acad Sci USA 2005; 102(32):11408-11413. 17. Kawamoto H, Katsura Y. A new paradigm for hematopoietic cell lineages: revision of the classical concept of the myeloid-lymphoid dichotomy. Trends Immunol 2009; 30(5):193-200. 18. Kincade PW, Owen JJ, Igarashi H et al. Nature or nurture? Steady-state lymphocyte formation in adults does not recapitulate ontogeny. Immunol Rev 2002; 187:116-125. € ƒ @ž    *    #^  !   $ ‚! Immunol 2006; 24:705-738. _• ‚% `‚   ! <          $   #   lineage. Science 1996; 271(5250):818-822. 21. Du T, Friend DS, Austen KF et al. Tissue-dependent differences in the asynchronous appearance of mast          ^    $      %  Clin Exp Immunol 1996; 103(2):316-321. 22. Furitsu T, Saito H, Dvorak AM et al. Development of human mast cells in vitro. Proc Natl Acad Sci USA 1989; 86(24):10039-10043. 23. Kempuraj D, Saito H, Kaneko A et al. Characterization of mast cell-committed progenitors present in human umbilical cord blood. Blood 1999; 93(10):3338-3346. 24. Ochi H, Hirani WM, Yuan Q et al. T helper cell type 2 cytokine-mediated comitogenic responses and CCR3 expression during differentiation of human mast cells in vitro. J Exp Med 1999; 190(2):267-280. 25. Kirshenbaum AS, Goff JP, Semere T et al. Demonstration that human mast cells arise from a progenitor cell population that is CD34( ), c-kit( ) and expresses aminopeptidase N (CD13). Blood 1999; 94(7):2333-2342. 26. Kirshenbaum AS, Kessler SW, Goff JP et al. Demonstration of the origin of human mast cells from CD34

bone marrow progenitor cells. J Immunol 1991; 146(5):1410-1415. 27. Mwamtemi HH, Koike K, Kinoshita T et al. An increase in circulating mast cell colony-forming cells in asthma. J Immunol 2001; 166(7):4672-4677.

MAST CELL PROGENITOR TRAFFICKING AND MATURATION

27

28. Eklund KK, Ghildyal N, Austen KF et al. Mouse bone marrow-derived mast cells (mBMMC) obtained in !     #    ^   !! "  #     $    †

@ and serosal mast cells from their normal littermates. J Exp Med 1994; 180(1):67-73. 29. Gurish MF, Ghildyal N, McNeil HP et al. Differential expression of secretory granule proteases in mouse mast cells exposed to interleukin 3 and c-kit ligand. J Exp Med 1992; 175(4):1003-1012. 30. Tsai M, Takeishi T, Thompson H et al. Induction of mast cell proliferation, maturation and heparin synthesis by the rat c-kit ligand, stem cell factor. Proc of Natl Acad Sci USA 1991; 88(14):6382-6386. 31. Galli SJ, Iemura A, Garlick DS et al. Reversible expansion of primate mast cell populations in vivo by stem cell factor. J Clin Invest 1993; 91(1):148-152. Œ_  #$ >  &           ˜&^#™     ^ arthritis. J Exp Med 2007; 204(12):2797-2802. 33. Hallgren J, Jones TG, Abonia JP et al. Pulmonary CXCR2 regulates VCAM-1 and antigen-induced recruitment of mast cell progenitors. Proc Natl Acad Sci USA 2007; 104(51):20478-20483. 34. Drew E, Huettner CS, Tenen DG et al. CD34 expression by mast cells: of mice and men. Blood 2005; 106(5):1885-1887. Œ  % =  < ` @##    @Œ’      <  $  $    =" ` 2002; 30(10):1211-1218. 36. Schrader JW, Lewis SJ, Clark-Lewis I et al. The persisting (P) cell: histamine content, regulation by a T-cell-derived factor, origin from a bone marrow precursor and relationship to mast cells. Proc Natl Acad Sci USA 1981; 78(1):323-327. 37. Gurish MF, Tao H, Abonia JP et al. Intestinal mast cell progenitors require CD49dbeta7 (alpha4beta7  ™  $^  # > ="  _••– €’˜€™_’Œ^__ 38. Abonia JP, Austen KF, Rollins BJ et al. Constitutive homing of mast cell progenitors to the intestine depends on autologous expression of the chemokine receptor CXCR2. Blood 2005; 105(11):4308-4313. 39. Sonoda T, Ohno T, Kitamura Y. Concentration of mast-cell progenitors in bone marrow, spleen and blood of mice determined by limiting dilution analysis. J Cell Physiol 1982; 112(1):136-140. 40. Gurish MF, Bell AF, Smith TJ et al. Expression of murine beta 7, alpha 4 and beta 1 integrin genes by rodent mast cells. J Immunol 1992; 149(6):1964-1972. 41. Yuan Q, Jiang WM, Hollander D et al. Identity between the novel integrin beta 7 subunit and an antigen found highly expressed on intraepithelial lymphocytes in the small intestine. Biochem Biophys Res Commun 1991; 176(3):1443-1449. 42. Boyce JA, Mellor EA, Perkins B et al. Human mast cell progenitors use alpha4-integrin, VCAM-1 and \ ƒ^ =^  #!   %# #$ !$ #$ $ {%  Blood 2002; 99(8):2890-2896. 43. Berlin C, Berg EL, Briskin MJ et al. Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell 1993; 74(1):185-195. ’’    `$#  ~= \ @   †”  ^    $<  $  attenuated protective immunity in the small intestine during enteric helminth infection. Eur J Immunol 2000; 30(6):1656-1664. 45. Kitamura Y, Yokoyama M, Sonoda T et al. Different radiosensitivities of mast-cell precursors in the bone marrow and skin of mice. Rad Res 1983; 93(1):147-156. ’“ `  > $ #  \#%  $    !    <  < #   routes of the mast cell. Immunol Rev 2007; 217:8-18. 47. Reutershan J, Morris MA, Burcin TL et al. Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung. J Clin Invest 2006; 116(3):695-702. ’— #< š ‚#$  ƒ \   ‚ ^_^   < $  # %   to initiate V(D)J rearrangement. Cell 1992; 68(5):855-867. 49. Alcaide P, Jones TG, Lord GM et al. Dendritic cell expression of the transcription factor T-bet regulates mast cell progenitor homing to mucosal tissue. J Exp Med 2007; 204(2):431-439. • & > # >& ƒ$^ž    *    *^ $ {   #  through its function in dendritic cells. J Clin Invest 2006; 116(2):414-421. 51. Rosenkranz AR, Coxon A, Maurer M et al. Impaired mast cell development and innate immunity in Mac-1 ˜@@— @‚Œ™^   > $ €€—– “˜_™“’“Œ^“’“” 52. Martelli F, Ghinassi B, Lorenzini R et al. Thrombopoietin inhibits murine mast cell differentiation. Stem Cells 2008; 26(4):912-919. 53. Ghinassi B, Zingariello M, Martelli F et al. Increased differentiation of dermal mast cells in mice lacking the Mpl gene. Stem Cells Dev 2009; 18(7):1081-1092. 54. Ammit AJ, Bekir SS, Johnson PR R et al. Mast cell numbers are increased in the smooth muscle of human sensitized isolated bronchi. Am J Respir Crit Care Med 1997; 155(3):1123-1129.  † # @= †  \     ^      % # $  # N Engl J Med 2002; 346(22):1699-1705.

28

MAST CELL BIOLOGY

56. Zanini A, Chetta A, Saetta M et al. Chymase-positive mast cells play a role in the vascular component of airway remodeling in asthma. J Allergy Clin Immunol 2007; 120(2):329-333. 57. Ikeda RK, Miller M, Nayar J et al. Accumulation of peribronchial mast cells in a mouse model of ovalbumin   $ #   % { $  $$  ~ ‹$ J Immunol 2003; 171(9):4860-4867. 58. Yu M, Tsai M, Tam SY et al. Mast cells can promote the development of multiple features of chronic asthma in mice. J Clin Invest 2006; 116(6):1633-1641. 59. Dillon SB, MacDonald TT. Limit dilution analysis of mast cell precursor frequency in the gut epithelium of normal and Trichinella spiralis infected mice. Parasite Immunol 1986; 8(5):503-511. 60. Abonia JP, Hallgren J, Jones T et al. Alpha-4 integrins and VCAM-1, but not MAdCAM-1, are essential   $        # { $ † _••“– •—˜™——^€’ “ # † #%  *$ @    ^      !$ {  $  % $       % { @ =" $ _••– ’˜_™__Œ^__€ 62. Gonzalo JA, Lloyd CM, Kremer K L et al. Eosinophil recruitment to the lung in a murine model of allergic { *#   *^ #<  #  > @ ! €€“– €—˜•™_ŒŒ_^_Œ’ 63. Briskin M, Winsor-Hines D, Shyjan A et al. Human mucosal addressin cell adhesion molecule-1 is preferentially expressed in intestinal tract and associated lymphoid tissue. Am J Pathol 1997; 151(1):97-110. 64. Xu B, Wagner N, Pham LN et al. Lymphocyte homing to bronchus-associated lymphoid tissue (BALT) is mediated by L-selectin/PNAd, alpha4beta1 integrin/VCAM-1 and LFA-1 adhesion pathways. J Exp Med 2003; 197(10):1255-1267. 65. Oliveira SH, Lukacs NW. Stem cell factor and igE-stimulated murine mast cells produce chemokines (CCL2, @@ƒ” @@ƒ__™  "  #<   { ‚ _••– •˜Œ™“—^”’ 66. Taub D, Dastych J, Inamura N et al. Bone marrow-derived murine mast cells migrate, but do not degranulate, in response to chemokines. J Immunol 1995; 154(5):2393-2402. 67. Collington SHJ, Pease JE, Jones T et al. The role of the CCL2/CCR2 axis in mouse mast cell migration in vitro and in vivo. Re-submitted to J Immunol 2010. 68. Weller CL, Collington SJ, Hartnell A et al. Chemotactic action of prostaglandin E2 on mouse mast cells acting via the PGE2 receptor 3. Proc Natl Acad Sci USA 2007; 104(28):11712-11717. 69. Weller CL, Collington SJ, Brown JK K et al. Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors. J Exp Med 2005; 201(12):1961-1971. 70. Jones TG, Hallgren J, Humbles A et al. Antigen-induced increases in pulmonary mast cell progenitor numbers depend on IL-9 and CD1d-restricted NKT cells. J Immunol 2009; 183(8):5251-5260. 71. Noben-Trauth N, Shultz LD, Brombacher F et al. An interleukin 4 (IL-4)-independent pathway for CD4

*^ ƒ^’  $  !  ƒ^’  ^   \  ~   ' €€”– 94(20):10838-10843. 72. Jankovic D, K Kullberg MC, Noben-Trauth N et al. Single cell analysis reveals that IL-4 receptor/Stat6 signaling is not required for the in vivo or in vitro development of CD4 lymphocytes with a Th2 cytokine    > $ _•••– “’˜“™Œ•’”^Œ• 73. Akbari O, Faul JL, Hoyte EG et al. CD4 invariant T-cell-receptor natural killer T-cells in bronchial asthma. N Engl J Med 2006; 354(11):1117-1129. 74. Lisbonne M, Diem S, de Castro Keller A et al. Cutting edge: invariant V alpha 14 NKT cells are required for  ^$  % {  # !   "  #  > $ 2003; 171(4):1637-1641. 75. Demoulin JB, Uyttenhove C, Van Roost E et al. A single tyrosine of the interleukin-9 (IL-9) receptor is required for STAT activation, antiapoptotic activity and growth regulation by IL-9. Mol Cell Biol 1996; 16(9):4710-4716. 76. Yoshimoto T, Min B, Sugimoto T et al. Nonredundant roles for CD1d-restricted natural killer T-cells and conventional CD4 T-cells in the induction of immunoglobulin E antibodies in response to interleukin 18 treatment of mice. J Exp Med 2003; 197(8):997-1005. 77. Madden KB, Urban JF Jr, Ziltener HJ et al. Antibodies to IL-3 and IL-4 suppress helminth-induced intestinal mastocytosis. J Immunol 1991; 147(4):1387-1391. ”— *% >   \ #% >   ƒ^€^   # $   ƒ^€ in pulmonary mastocytosis and goblet cell hyperplasia but not T-cell development. Immunity 2000; 13(4):573-583. ”€ # @†  # => @ †   = {$ # $  $  $    $     $    $  { > $ _••€– —_˜’™_’“^_’_’ 80. Urban JF Jr, Noben-Trauth N, Schopf L et al. Cutting edge: IL-4 receptor expression by nonbone marrow-derived cells is required to expel gastrointestinal nematode parasites. J Immunol 2001; 167(11):6078-6081.

CHAPTER R3 MONOMERIC IgE AND MAST CELL $'$( $%& )*'+' % ^)%&+(% >$^# #%<$     ˆ  *#< %<| Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA |   !" #$" 

Abstract:

Mast cells play a major role in allergy and anaphylaxis, as well as a protective role in immunity against bacteria and venoms (innate immunity) and T-cell activation (acquired immunity).1,2 It was long thought that two steps are essential to mast cell ! *#   ˜}™ $  %# ^  =    ##^  =  ˜¡RI) expressed on the surface of mast cells. The second  $  %#  ˜™  ^=  ^  =  bound to Fc¡RI present on the mast cell surface (this mode of stimulation hereafter referred to as IgE Ag or IgE anti-IgE stimulation, respectively). Conventional wisdom has been that monomeric IgE plays only an initial, !     ! `%!    #! #% # IgE binding to its receptor Fc¡RI can mediate mast cell activation events even in the absence of antigen (this mode of stimulation hereafter referred to as IgE(-Ag) stimulation). Different subtypes of monomeric IgEs act via IgE(-Ag) stimulation to elicit varied effects on mast cells function, survival and differentiation. This chapter will describe the role of monomeric IgE molecules in allergic reaction, the various effects and mechanisms of action of IgE(-Ag) stimulation on mast cells and what possible developments may arise from this knowledge in the future. Since mast cells are involved in a variety of pathologic and protective responses, understanding the role that monomeric IgE plays in mast cell function, survival and differentiation will hopefully lead to better understanding and treatment of asthma and other allergic diseases, as well as improved understanding of host response to infections.

INTRODUCTION Over the past few decades, the number of patients with allergic diseases (e.g., asthma, atopic dermatitis) has risen markedly in industrialized nations. Thirty-nine percent of Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 29

30

MAST CELL BIOLOGY

children and 30% of adults have been diagnosed with one or more of asthma, eczema and hay fever.3 Allergic diseases are manifested by an immune system over-reaction to a substance that the body should not normally identify as dangerous and that reaction can be something as basic as a mild irritation or it can be so extreme as to cause fatal results. The mast cell is a type of myeloid lineage hematopoietic cell and its development and function suggest that it is involved heavily in the pathogenesis of these allergic diseases (e.g., when a mast cell is “activated” it releases chemicals into the body that cause what we call an allergic reaction). Mast cells originate from precursor cells in the bone marrow, enter the circulation and then localize to mucosal and epithelial sites under # {$  #   ! 4 At these sites, facing the outer environment and surrounding blood vessels, mast cells can produce and release a vast array of biogenic amines (e.g., histamine and serotonin), lipids (e.g., leukotrienes and prostaglandins), nucleotides (e.g., adenosine) and polypeptides (e.g., proteases, cytokines and chemokines) #    $     {    %    adaptive immune responses.5,6 Indeed, mast cells from atopic individuals that are sensitized   ^  $$ = ˜=™        cell-derived mediators. Subsequent allergen exposure in susceptible individuals produces a cascade of events orchestrated by immune effector cells such as Type 2 helper T (Th2) cells and eosinophils.7 B cells stimulated by IL-4 or IL-13 produce IgE.8 Recent studies have shown that monomeric IgE can activate mast cells in the absence of antigen. This suggests that allergic symptoms may be due in part to the effects of monomeric IgE and that these symptoms may occur even in patients who are not exposed to allergens. Different subtypes of IgE have been shown to have different effects on mast cells. It is conceivable that different types of monomeric IgE may give rise to the range of symptoms and symptom severity observed amongst allergic patients. Anti-IgE therapy #   $     %#      #   role IgE must have in asthma and other allergic diseases.9 It is still unknown how anti-IgE therapy relieves allergic symptoms, but it is highly possible that preventing the effects of monomeric IgE on mast cells is a large part of how anti-IgE therapy mediates its effects. Elucidating the true role and mechanism of action of monomeric IgE’s effects on mast cells will further our understanding of allergic diseases, symptoms and treatment. This chapter will review what is known about the interaction between monomeric IgE and mast cells in the absence of antigen. IgE Ag STIMULATION OF MAST CELLS The initial step in IgE  !$  %# ^  IgE binds Fc¡RI, stabilizing and increasing the number of Fc¡RI receptors on the mast cell surface.10 *# } #         # #   can be activated immediately upon subsequent antigen exposure. Antigen binding to ^  ¡RI-bound, IgE cross-links Fc¡RI (Fc¡RI aggregation/cross-linking), thereby inducing various mast cell activation events (e.g., degranulation, de novo synthesis     {    $ !!     ™ ! $ signal transduction pathways within the mast cell. In response to Fc¡RI cross-linking, various phosphorylation cascades are triggered,   $ {$" $           degranulation, eicosanoid generation and chemokine/cytokine production. The Fc¡RI

MONOMERIC Ig$ % & $ $'$( $%& )*'+' ' % ^)%&+(%

31

receptor consists of four subunits (_`a2)—one _ subunit that binds IgE, one ` subunit !!      ¡‚ }  % $ ^ a subunits acting as the main signal transducer.10 Fc¡RI aggregation leads to the activation of Lyn, a Src family protein tyrosine kinase (PTK) associated with the ` subunit of Fc¡RI. The intracellular parts of Fc¡RI ` and a contain immunoreceptor tyrosine-based activation motifs, or ITAMs, that consist of six conserved amino acid residues spread out over an ¾26 amino acid sequence, (D/E)X7(D/E)X2YX2(L/I)X6-8YX2(L/I).11 Activated Lyn phosphorylates tyrosine residues in these intracellular Fc¡RI ` and a subunit ITAMs. ` phosphorylated ITAM recruits Lyn PTK K and a phosphorylated ITAM recruits Syk PTK. Two other Src family PTKs, Fyn and Hck, also work with Lyn and Syk to activate several signaling pathways, especially the PI3K K signaling pathway.12 Signaling events activated by these PTKs (PI3K, phopholipase C/Ca2 and several MAPK K pathways) lead to cytokine/chemokine production and degranulation of the effector molecules mentioned in the introduction (chemicals, lipids, nucleotides and polypeptides).13,14

IgE(-Ag) STIMULATION OF MAST CELLS: MONOMERIC IgE AFFECTS MAST CELL FUNCTION IN THE ABSENCE OF ANTIGEN As mentioned above, monomeric IgE appears to be involved in more than just mast cell sensitization. IgE(-Ag) induces up-regulation of Fc¡RI expression on the cell surface,15-17 Fc¡RI stabilization,18,19 increased histamine content,20 histamine and leukotriene release, receptor internalization, cytokine/chemokine expression,21 increased response to compound 48/80 and substance P,22 F-actin content release,23 membrane ${24   #25 and migration.26 Almost all of these effects are seen with IgE Ag and IgE anti-IgE stimulation as well (Fig. 1). However, in IgE(-Ag) stimulation, these effects are seen at IgE concentrations 2-3 logs higher than what are required for Ag-dependent IgE Ag mast cell activation. Since IgE(-Ag), IgE Ag and IgE anti-IgE can mediate similar effects at the subcellular level, it follows that IgE(-Ag) would be able to elicit mast cell responses similar to those seen with IgE Ag and IgE anti-IgE. Indeed, IgE(-Ag) has been shown to (i) promote mast cell survival, (ii) amplify mast cell accumulation at allergic tissue sites and (iii) mediate mast cell activation events. (i) IgE(-Ag) stimulation seems to play a role in preventing mast cell apoptosis. Mast cells undergo apoptosis at low levels of growth factor both in vitro and in vivo. Two groups have investigated whether monomeric IgE can inhibit the mast cell apoptosis that is induced by the removal of growth factor; their results showed that increasing the concentration of monomeric IgE can indeed prevent mast cell apoptosis.18,27 IgE(-Ag), IgE anti-IgE and IgE Ag (at low antigen concentrations) stimuli of Fc¡RI all promote mast cell survival.28 The duration and strength of this Fc¡RI stimulation determines mast cell survival (further discussed under Signal Transduction induced by Monomeric IgE). (ii) IgE(-Ag) and IgE          # {  response. Epicutaneous application of IgE(-Ag) and IgE Ag at allergic tissue sites with local high IgE levels induces mast cell accumulation.29 Mast cell accumulation can amplify {   ! $ $#  # $<  †4 (LTB4) and chemokines/cytokines released during mast cell degranulation. These effector molecules recruit other immune cells such as T cells, eosinophils, monocytes and neutrophils and $ #  #  ^{   $ ` # # secretion of Th2 cytokines (involved in mediating allergic responses and host defense

32

MAST CELL BIOLOGY

Figure 1. Biological effects by various modes of stimulation via the Fc ¡RI. Experiments using mouse BMMCs are summarized. Concentrations of stimuli are as follows: PC IgE (H1 DNP-¡-206) and HC IgE (SPE-7), 0.5 +g/ml (low) and 5 +g/ml (high); and IgE Ag (DNP21-BSA), 1 ng/ml (low) and 100 ng/ml (high). Notice that biological events are listed in a rough order of occurrences and receptor aggregation presumably occurs in all modes of stimulation, except for low PC IgE concentrations. –, not detected, -#, very weak; W, weak; W# weak¾moderate; S#, moderate¾strong; and S, strong. Reproduced with permission from: Kawakami T, Kitaura J. J Immunol 2005; 175:4167-4173;28 ©2005 The American Association of Immunologists, Inc.

against parasitic infections)30 and inhibits the production of Th1 cytokines.31 LTB4 recruits T cells and myeloid cells.32-34 Transcripts coding for the CC chemokines (chemokines in %## #  %  $   ‘™35 I-309/CCL1, MIP-1_/CCL3, MIP-1`/ CCL4 and MCP-3/CCL7 are dramatically enhanced in IgE Ag-stimulated mast cells.36 These cytokines and chemokines recruit T cells, eosinophils, monocytes and neutrophils. Activated mast cells may also release mediators to further induce activation of themselves and their neighboring mast cells. Adenosine and sphingosine 1-phosphate acutely released by IgE Ag play an important role in Ca2 {$"  $  chemotaxis to antigen.37,38 PTK-based signaling pathways and G-protein coupled receptor (GPCR)-based signaling pathways dependent on phosphoinositide 3-kinase a (PI3K Ka) cooperate to enhance degranulation and migration. It is likely that they induce other activation events as well. Factors that bind to GPCRs (sphingosine 1-phosphate, adenosine, LTB4 and chemokines MIP-1_/CCL3, MIP-1`/CCL4, MIP-2/CXCL1-3, MCP-1/CCL2 and RAN A TES/CCL5) contribute to IgE(-Ag) and IgE Ag mast cell migration using both directional (chemotaxis) and nondirectional (chemokinesis) mechanisms.26 *#     =    {^$    <       {    $ !  # removal of antigen as long as local IgE synthesis continues. A positive feed-back-loop ## %    " {     %# ##  $ IgE levels.14 B IgE A B Fc¡RI, B mast cells A B Ag-IgE and Fc¡RI-dependent release of IL-4, IL-13, MIP-1_/CCL3 and so on A B IgE. (iii) IgE(-Ag) stimulation exerts a vast number of effects on mast cells that all play a role in the activation of human as well as murine mast cells. For instance, IgE(-Ag) induces the production of various effector molecules. Monomeric IgE induces production

MONOMERIC Ig$ % & $ $'$( $%& )*'+' ' % ^)%&+(%

33

  ^{  ƒ^—   #  ^ ˜ @\^™    derived from umbilical cord blood.39   <%    # {   inhibit production of these chemokines. In human lung mast cells, monomeric IgE not only induces IL-8 production, but also induces mast cell degranulation, LTB4 production   $ $ {$"40 Mast cells derived from human umbilical cord blood synthesize and release I-309/CCL1 (a ligand for CCR8 that chemoattracts Th2 cells), GM-CSF and MIP-1_/CCL3 in response to IgE(-Ag) stimulation.41 IgE(-Ag) also activates mast cells by increasing the number of Fc¡RI on the mast cell surface. Although increased numbers of Fc¡RI are also seen after IgE Ag stimulation, IgE(-Ag) increases Fc¡RI numbers via receptor stabilization while IgE Ag does this via actual upregulation of Fc¡RI expression.42,43 Furthermore, in the case of IgE Ag, the number of Fc¡RI on the mast cell surface may actually decrease at very high levels of Ag due to receptor internalization.44 Microarray analysis of 760 human genes showed changes in mast cell gene expression indicative of mast cell activation by IgE(-Ag) stimulation. Fifty-eight genes are upregulated after IgE(-Ag) stimulation and a total of 115 genes are upregulated during IgE anti-IgE stimulation. The transcripts upregulated by IgE(-Ag) include many that are expected for immune cell activation: genes coding for cytokines (IL-1`, IL-6 and CSF1), chemokines (IL-8/CXCL8, MIP-1`/CCL4, MCP3/CCL7, GRO_/CXCL1 and GROa/CXCL3), cytokine and chemokine receptors, regulators of signal transduction and transcription factors, as well as other genes involved in immune regulation, cell proliferation and survival, adhesion and cytoskeletal remodeling.45  $  #$ $$    %# =     # expression of two nitric oxide (NO) generating enzyme isoforms, nitric oxide synthase 2 and 3 (NOS2 and NOS3, respectively). Interestingly, IgE anti-IgE stimulation leads to further upregulation of NOS2 expression but decreased NOS3 expression.46 This is  %#    #    %#  #  $ NOS3 expression 30 minutes after antigen exposure followed by a subsequent increase in NOS2 expression concomitant with increased airway reactivity.47,48 IgE-mediated activation phenomena have also been observed in Fc¡RI-expressing cells other than mast cells. For instance, monomeric IgE stimulates basophils (which are Fc¡RI-expressing cells) to produce CCL22 (macrophage-derived chemokine, or MDC), a chemoattractant for Th2 cells.49 Monomeric IgE also inhibits apoptosis of neutrophils, which express Fc¡RI in atopic asthmatics and are thought to play a role in the development of asthma.50 Activation of extracellular signal-related kinase (ERK), a required step for IgE(-Ag)-mediated mast cell survival, is required for differentiation of immature B cells into transitional and mature B cells.51   #     #$# #  #   #$   IgE exerts various effects upon cells that express Fc¡RI, even in the absence of antigen.

MONOMERIC IgE SUBTYPES (HIGHLY CYTOKINERGIC IgE VS POORLY CYTOKINERGIC IgE) Monomeric IgEs vary in their ability to induce different IgE(-Ag) mast cell activation events. This was brought to light by the inconsistencies found between the characteristics of monomeric IgE observed in initial studies done by K Krystal et al and our research group. For instance, we found that monomeric IgE prolonged mast cell survival, but did

34

MAST CELL BIOLOGY

not affect mast cell proliferation, phosphorylation of signaling molecules, production of various cytokines and expression of apoptosis inhibitors. However, K Krystal et al were able to induce almost all these mast cell activation phenomena. Another group found that monomeric IgE can induce actin polymerization.23 It was thought that these discrepancies might have been due to a difference in the monomeric IgEs used in our experiments and various IgE molecules were examined with regards to their effect on mast cell activation and survival. As a result, it was shown that certain types of IgE molecules (highly cytokinergic IgE: HC IgE) not only prolong mast cell survival but also strongly {$    $        26 and adhesion   29 leukotriene release, production of chemokines/cytokines such as IL-6 and TNF-_, phosphorylation of signaling molecules, as well as Fc¡RI aggregation21 and internalization.44 On the other hand, there exist types of IgE molecules (poorly cytokinergic IgE: PC IgE) that do not affect mast cell activation processes other than prolongation of mast cell survival.52 In this manner, it was shown that individual IgE molecules have different properties (Fig. 1). There is some knowledge about the differences underlying the divergent effects of HC and PC IgE on mast cell degranulation at a molecular level. Mast cell degranulation involves fusion of the secretory granule membrane with the plasma membrane. This membrane fusion reaction is mediated by a group of integral membrane proteins called SNAREs.53 SNAREs localize to detergent-insoluble lipid raft microdomains in Fc¡RI-stimulated mast cells. There are two main categories of SNAREs, vesicle SNAREs (or v-SNAREs) present on vesicle membranes in the vesicle-associated membrane protein (VAMP)/ synaptobrevin family and target SNAREs (or t-SNAREs) present on target membranes in the syntaxin and SNAP-23 family. The majority of SNAP-23 is lipid raft-associated, while only small amounts of syntaxin-4 or VAMP-2 are raft-associated. One copy each of SNAP-23/syntaxin/VAMP must form a ternary complex in lipid raft membranes for membrane fusion to occur.54 HC IgE seems to regulate VAMP-2 expression at the level of mRN R A (transcriptional and/or posttranscriptional). Monomeric IgE also seems to play a role in determining the subcellular location of SNAREs. SNAP-23 and VAMP-8 protein levels were preferentially localized to lipid rafts in mast cells cultured with HC IgE and high concentrations of PC IgE when compared to mast cells cultured with low concentrations of PC IgE.55 Still, a clear explanation for the marked heterogeneity among IgE molecules has yet to be uncovered, as no information is truly available about the molecular differences      =          = $¢ typical HC IgE, SPE-7 and a much weaker HC IgE, H1 DNP-¡-26, have similar reported   ˜  ¾15 nM).19,56 Anisotropy data has suggested that there is a difference in the extent of receptor aggregation between different IgE types. The more extensive receptor aggregation seen with HC IgE over PC IgE may help explain how a strong HC IgE can induce all activation events, whereas PC IgE can induce only a limited set of activation events to a lesser degree.21 The dichotomy between HC and PC IgEs is reminiscent of the variability between different human IgE molecules’ ability to prime basophils for stimulation with histamine-releasing factor (HRF). HRF is a cytokine produced by macrophages and platelets. Basophils bound by IgE derived from atopic patients release histamine and cytokines such as IL-4 and IL-13 in response to HRF.57,58 Basophils bound by IgE from normal individuals do not show this same response. IgE that has the ability to respond to HRF was termed IgE and IgE that does not have the ability to respond to HRF was

MONOMERIC Ig$ % & $ $'$( $%& )*'+' ' % ^)%&+(%

35

termed IgE<. There is no information about if or how structural differences may underlie the functional differences between IgE and IgE<. However, altered usages of heavy-chain variable (VH) genes have been reported in IgE of allergic patients. There is overrepresentation of VH5 (one of the smallest gene families containing two members out of the total of 52 functional VH genes) in IgE derived from allergic patients.59 This indicates that there is indeed structural heterogeneity between human IgE molecules. Further study of human IgE heterogeneity may reveal relevance to human atopic and allergic diseases.

IgE(-Ag) STIMULATION: HYPOTHESIZED MECHANISMS OF ACTION Questions remain about how IgE(-Ag) mediates mast cell responses similar to those seen after IgE Ag and IgE anti-IgE stimuli. It is established that IgE Ag or IgE anti-IgE requires Fc¡RI aggregation to mediate mast cell activation.60 This Fc¡RI aggregation mechanism is consistent with the common mechanism (receptor dimerization/ oligomerization) seen with receptor-mediated cell activation.61,62 Biophysical methods, including time-resolved phosphorescence anisotropy (which can assess the size and rigidity of membrane structures by quantifying, in the nano- to microsecond range, the motion of phosphorescent dye-labeled membrane proteins that are contained within the membrane), have suggested that IgE(-Ag) stimulation also results in Fc¡RI aggregation.52 Calculations have shown that IgE-IgE interactions on the cell surface (assumed to be Fc¡RI aggregation) took place at IgE concentrations 1 +g/mL in the absence of Ag. This is consistent with data found at several laboratories that IgE effects on survival and activation are seen at * 0.5 +g/mL IgE concentrations.28 IgE Ag stimulation requires localization of aggregated receptors in cholesterol/ sphingolipid-rich plasma membrane domains (lipid rafts).63,64 Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in # $   $     <54 Disruption of lipid rafts prevents IgE(-Ag) induced ERK K activation, suggesting that localization of aggregated receptors in lipid rafts is also necessary for IgE(-Ag) stimulation.19 #$# #  $ # ¡RI aggregation is required for IgE(-Ag) stimulation, it is still unclear how IgE could cross-link Fc¡RI in the absence of antigen. There are several models that can be formulated regarding the mechanism of cross-linking # $   #       #$# #      escape the realm of conjecture. Since monovalent hapten binds to the variable (V) region and inhibits monomeric IgE effects, the V-region of monomeric IgE is likely involved somehow in mediating those IgE(-Ag) effects (such as receptor aggregation). For instance, a monomeric IgE molecule may bind to Fc¡RI and then use itself or another molecule to mediate Fc¡RI cross-linking in an Ig-Ig or Ig-X-Ig (X  unknown bridging molecule) type interaction. One type of HC IgE in particular, SPE-7 IgE, can change the initial structure of its variable region and then bind different antigens, inducing isomerization  #      ##^  " %#     %  site.65 It is possible that the fragment antigen-binding (Fab) portion of Fc¡RI-bound IgE directly or indirectly interacts with a neighboring Fc¡RI-bound IgE via a third molecule that  }  # #  = $ #      # \=#^ expressed by Schistosoma mansoni binds to IgE,66 it is thought that proteins derived from parasites and other organisms cross-link monomeric IgE. Studies have also shown that a 28 kDa protein, p28, may be involved in the Fc¡RI aggregation mediated by monomeric

36

MAST CELL BIOLOGY

IgE. p28 is associated with ¾50% of Fc¡RI in RBL-2H3 rat mast cells in the absence of IgE, but dissociates from Fc¡RI at a high concentration (20 +M) of IgEs.67 The identity of this p28 sensor protein is still unknown and further studies must be done to reveal the true involvement of p28 in IgE(-Ag) receptor aggregation. Despite the building evidence that IgE(-Ag) effects are due to binding by monomeric, not aggregate, IgE molecules, it still needs to be conclusively proven that IgE does not simply aggregate before binding Fc¡RI at concentrations that result in IgE(-Ag) Fc¡RI aggregation.28 The exact events that take place after IgE(-Ag)-induced Fc¡RI aggregation also remains to be understood. Currently, it is thought that cytokines such as IL-3 and IL-6 act in an autocrine fashion on mast cells following IgE(-Ag) stimulation. Pro-/anti-apoptotic Bcl-2 family member proteins and mitogen-activated protein kinases (MAPKs) seem to play a role in mediating the effects of IgE(-Ag). Signaling via toll-like receptors (TLRs) synergistically augment the effects of IgE(-Ag) stimulation, likely by modulating the expression of Bcl-2 family member proteins and phosphorylating MAPKs.68 !   $  #   # `@ =  $   of cytokine(s) that support mast cell survival in an autocrine/paracrine manner. Fc¡RI aggregation appears to induce autocrine secretion of IL-3, activating IL-3R R signaling pathways that promote survival.69 Stat5 was found to be an important part of this activated IL-3R R signaling pathway.70 ~$ }  ƒ^Œ  ƒ^Œ     $ but did not completely prevent, HC IgE-mediated survival effects.69 Furthermore, Krystal K et al reported that monomeric IgE requires phosphorylation of ERK K and intracellular $ {$" %#   $ #% $   $  ! oxygen species (ROS) that induce IL-3 production.71 The cytokine IL-6 also seems involved in mast cell survival. Adding IgE to human lung mast cells (HLMC) leads to increased HLMC IL-6 mRN R A expression and protein release. Anti-IL-6 antibody, but not anti-SCF antibody, prevents IgE(-Ag) mast cell survival effects. This suggests that IL-6 plays an important role in mediating IgE(-Ag) mast cell survival effects. This is consistent with the knowledge that IL-6 prevents apoptosis in HLMC and cord blood-derived mast cells (HCBMC).72,73 Bcl-2 family members play a key role in mediating IgE(-Ag)-induced mast cell survival. Expression of anti-apoptotic Bcl-2 family members, Bcl-xL and Bcl-2, is dependent on IL-3 and it has been reported that HC IgE prolongs mast cell survival by autocrine IL-3 production to increase the expression of Bcl-xL.69 In line %# #  †^"ƒ  $    $ !!  ƒ^Œ
MONOMERIC Ig$ % & $ $'$( $%& )*'+' ' % ^)%&+(%

37

combinations of HC IgE with LPS or PGN. However, while JNK inhibitor (SP600125) and ERK inhibitor (U0126) inhibit mast cell response to HC IgE, they have little effect on mast cell response to TLR ligands (LPS, PGN) and only partially inhibit response to combinations of HC IgE with LPS or PGN.74 Thus, MAPKs are involved in IgE(-Ag)-mediated mast cell activation, but much more must be elucidated about their exact role in IgE(-Ag) activation pathways. Interestingly, PC IgEs do not support survival of Fc¡RI_
SIGNAL TRA R NSDUCTION INDUCED BY MONOMERIC IgE In mast cells, Fc¡RI aggregation induces phosphorylation of ITAMs in ` and a subunits of Fc¡‚     ! # $# \* ˜ƒ  `<  Syk), LAT (linker for activation of T cells) and SLP-76. Activation of these signaling pathways induces mast cell degranulation and cytokine production.78,79 At all levels of the Fc¡RI signaling cascade, both subtle and major similarities and differences are being revealed about how IgE(-Ag) and IgE Ag are unique. For instance, HC IgE(-Ag) signaling induces phosphorylation of the Fc¡RI ` subunit in a manner similar to IgE Ag.80 However, the phosphorylation kinetics of signaling molecules differ between the two types of stimulation.26,27 Also, phosphorylation of unique sets of signaling molecules are differentially required for the induction of various mast cell activation events. Differences have been found between the roles of PKC` I and II in IgE(-Ag) and IgE Ag stimulation of bone marrow mast cells.52,81,82 IgE(-Ag)-induced mast cell migration relies on the Lyn, Syk and Fyn PTKs and protein kinase C (PKC) `, ¡ and e to a lesser degree.26 IgE(-Ag)-mediated survival does not seem to require Fyn, Gab2, PI3K K p85, or Akt.69

  #    $} ƒ  < $   \*  %  # Src family kinases and PKCe.29 The Fc¡RI a subunit (FcR Ra) ITAM is required in different ways for various mast cell functions. Also, the FcR Ra-ITAM is utilized differently by IgE(-Ag) and IgE Ag to mediate the same mast cell function.83 IgE(-Ag) requires the _ and a subunits of Fc¡RI to mediate mast cell survival.18,84 IgE(-Ag)-induced cytokine production is FcR Ra-ITAM-dependent, but IgE(-Ag)-induced upregulation of Fc¡RI is FcR Ra-ITAM-independent. IgE(-Ag)-induced receptor up-regulation also does not require Lyn, Syk, or other PTKs that are required by degranulation and other activation events.44 The differences in Fc¡RI subunits and

38

MAST CELL BIOLOGY

signaling molecules utilized by IgE(-Ag)-induced Fc¡RI up-regulation is consistent with the observation (mentioned earlier in IgE(-Ag) Stimulation of Mast Cells (iii)) that IgE(-Ag)-induced Fc¡RI up-regulation is due more to the increased lifetime of surface-resident receptors rather than to its increased synthesis and/or transport to the plasma membrane.15,42,43 It seems that the duration and strength of Fc¡RI signaling determines the type of mast cell response. Activation of ERK K by activated Lyn and Syk is part of the initial Fc¡RI signaling pathway.21 It is known that the duration of ERK K activation alters the quality of biological responses (sustained ERK K activation is required for mast cell survival but not degranulation).85,86 The prolonged activation of ERK K seen with slow on-rate IgE interaction with Fc¡RI is in line with the pattern of ERK K activation required for survival of other cell types such as T cells.87 The Fc¡RI a subunit signaling required for survival of CD8//a-expressing a
channel inhibitors, La3 or Gd3 , do not. IgE Ag induced Ca2 responses are suppressed by all three inhibitors.90 This suggests that IgE(-Ag) and IgE Ag induce Ca2 {$" ! different mechanisms (Fig. 2). While IgE(-Ag) and IgE Ag both elicit mast cell responses via Fc¡RI signaling pathways that utilize similar sets of signaling molecules, key differences between the signaling pathways are being uncovered. Elucidating the similarities and differences between IgE(-Ag) and IgE Ag signaling will further our understanding of allergic disease at a cellular level.

MONOMERIC Ig$ % & $ $'$( $%& )*'+' ' % ^)%&+(%

39

Figure 2. Comparison of signaling events induced by different modes of Fc¡RI stimulation. Data with mouse BMMCs are summarized. Concentrations of stimuli are as follows: PC IgE (H1 DNP-¡-206) and HC IgE (SPE-7), 0.5 +g/ml (low) and 5 +g/ml (high); and IgE Ag (DNP21-BSA), 1 ng/ml (low) and 100 ng/ml (high). Notice that biological events are listed in a rough order of occurrences and receptor aggregation presumably occurs in all modes of stimulation, except for low PC IgE concentrations. –, not detected, -#, very weak; W, weak; W# weak¾moderate; S#, moderate¾strong; and S, strong. Reproduced with permission from: Kawakami T, Kitaura J. J Immunol 2005; 175:4167-4173;28 ©2005 The American Association of Immunologists, Inc.

IN VIVO EFFECTS OF IgE(-Ag) STIMULATION #$#         !  !! !  =˜^™^ mast cell survival and activation, supporting data have recently accumulated. Increased mast cell numbers were seen in mice transplanted with IgE-producing hybridomas that increased serum IgE levels.21 However, mast cell numbers were not increased in IgE-transgenic mice,91 and the presence of IgE was not found to be a requirement for mast cell development.92 *#   $ # = !    # <   #    $  !! `%!  #   not undermine the previously hypothesized positive effects that IgE(-Ag) may have on mast cell survival, since the concentration of IgE must be relatively high for the survival   =˜^™  <    !    $  =˜^™^ mast cell survival and activation. There is in vivo evidence that IgE may be needed to maintain mast cell survival during parasitic infections. In mice infected with Trichinella spiralis, monomeric IgE is needed to protect the survival of mast cells.93 In wild-type mice, Trichinella spiralis infection elicits an IgE response (pronounced expansion of the mast cell population in the intestine and spleen).93 In IgE
40

MAST CELL BIOLOGY

mice. This suggests that high-level IgE is essential in promoting mast cell survival in vivo by preventing mast cell apoptosis in the spleen. There is also in vivo support of the proposed IL-3 autocrine mechanism for mast cell survival induced by IgE(-Ag). In IL-3
MONOMERIC IgE AND MAST CELL DIFFERENTIATION Mast cells differentiate from hematopoietic progenitor cells in the bone marrow after progenitor cells migrate to local tissues.99-101 Stem cell factor (SCF) and its receptor, KIT, are essential for the differentiation, proliferation and survival of mast cells.102 However, other cytokines, growth factors and other factors are also involved in the development, proliferation, survival and differentiation of mast cells.103 Monomeric IgE acts not only upon mature mast cells, but works on mast cell progenitors to induce differentiation into mature mast cells. Bone marrow cells grown for 4 weeks in IL-3 culture supplemented with monomeric IgE, especially HC IgE, resulted in an increased number of mast cells compared with cultures grown without IgE. Also, although bone marrow cells cultured in IL-3-containing medium do eventually consist of 85% mast cells, the presence of IgE (especially HC IgE) reduces the time period required for the mast cell purity to reach *80%.55 Properties of mast cells exhibit heterogeneity, depending on what tissue and species they are derived from.104,105 Mast cells can exhibit differences in lifespan, morphology, development, expression pattern of mouse mast cell proteases (mMCPs) and proteoglycans and sensitivity to immunologic and nonimmunologic stimuli.106-111 Monomeric IgE binds the Fc¡RI receptor expressed on mast cell precursors in the early stages of mast cell differentiation to alter the expression of various genes, especially

MONOMERIC Ig$ % & $ $'$( $%& )*'+' ' % ^)%&+(%

41

those genes that are relevant to differentiation. As a result, different IgE subtypes can modulate the mast cell phenotype in various ways and likely play a role in giving rise to mast cell heterogeneity. For example, the expression of early growth response factor-1 (Egr-1) is upregulated in cultures containing high and low concentrations of HC IgE and a high concentration of PC IgE. Egr-1 is a transcription factor involved in the production of TNF-_ in mast cells. TNF-_ helps mediate IL-3-dependent differentiation of mast cells.112 TNF-_ expression was increased in bone marrow cells cultured with HC IgE and high concentrations of PC IgE (consistent with the increased Egr-1 expression seen with HC IgE and high concentrations of PC IgE). However, levels of TNF-_ protein were barely detectable even in bone marrow cells cultured with HC IgE. Expression of IL-13 and TGF-` is comparable in bone marrow cells cultured with different subtypes of IgE.55 *#  $ #   = $      plays a role in determining mast cell phenotype.

POLYCLONAL IgE AND MAST CELL ACTIVATION Although there is evidence that monoclonal IgE acts in an IgE(-Ag) manner to induce mast cell activation and mast cell differentiation, the effect of polyclonal IgE on mast cells has not been studied until recently. A mixture of equal amounts of several different types of monoclonal monomeric IgE promoted cell survival and induced cytokine production at the level that would be expected based on the average of the different types of monomeric IgE.21 This suggested that polyclonal IgE would also be able to affect mast cell survival and function. Polyclonal monomeric IgE taken from the serum of mice with atopic dermatitis induced by epicutaneous administration with mite antigen and Staphylococcus Enterotoxin B (SEB)113,114 resulted in prolonged mast cell survival and cytokine production in comparison to serum from mice without atopic dermatitis. This prolonged mast cell survival and cytokine production was not seen when IgE was removed from the blood or when the  $ $} %   =^   %#        cell survival and cytokine production was not seen when polyclonal monomeric IgE from the serum of mice with atopic dermatitis was added to mast cells from Fc¡RI_^  mice. These results suggest that polyclonal IgEs mediate their effects via Fc¡RI in a similar manner to monoclonal IgEs. The effect of human polyclonal monomeric IgE, from the serum of patients with atopic dermatitis, on human peripheral blood-derived cultured mast cells was additionally studied. IL-8 production was increased in mast cells cultured with serum taken from patients with atopic dermatitis when compared to mast cells cultured with serum added from healthy subjects. Removing IgE from the serum inhibited IL-8 production.115 Previous studies have dealt with the effects of monoclonal monomeric IgE. However, monomeric IgE in the serum of actual patients is polyclonal. The results of these polyclonal   = $        $ #  ! #  ! that polyclonal monomeric IgE in the serum of patients does actively stimulate mast cells. Better understanding the effects of polyclonal monomeric IgE is the next step in proving that the effects of monomeric IgE play an active role in the development of allergic diseases.

42

MAST CELL BIOLOGY

CONCLUSION Mast cells play a major role in allergy, anaphylaxis, innate immunity and acquired immunity. The role of monomeric IgE was previously thought to be limited to passive sensitization of the mast cell for IgE Ag activation. It is now understood that monomeric IgE plays a much larger role in mast cell function and can induce mast cell activation events via IgE(-Ag) stimulation. The mechanisms of action by which IgE mediates its effects on mast cells via IgE Ag and IgE(-Ag) stimulation are being uncovered. Fc¡RI aggregation seems to play a large role, as do the signaling molecules Lyn, Fyn, Hck and Syk. Evidence supports the role of autocrine IL-3 secretion in IgE(-Ag) stimulation. Experiments being done studying the differences between HC IgE and PC IgE suggest that different subtypes of monomeric IgEs elicit different sets of mast cell responses and that the different IgE subtypes may work via different mechanisms of action. While there   <   ! !  =˜^™   !! #  #!    studies, especially those looking at mast cell survival in mice with parasitic infections. The more recent studies have elucidated a role for IgE(-Ag) in mast cell differentiation in addition to its previously acknowledged role in mature mast cell survival and activation. The most recent studies have also found that polyclonal monomeric IgE has effects on mast cell function similar to those seen with monoclonal monomeric IgE stimulation. *#     #    #  #   =  a larger role in mast cell function, survival and differentiation than previously believed. Since the mast cell is involved in allergy, anaphylaxis and both innate and acquired immunity, better understanding of the effects and mechanisms of action of IgE(-Ag) stimulation will lead to an improved understanding of these pathologic and protective responses. This, in turn, may lead to the uncovering of better treatments for asthma and other allergic diseases, as well as ways to improve host response to infections.

REFERENCES 1. Galli SJ, Maurer M, Lantz CS. Mast cells as sentinels of innate immunity. Curr Opin Immunol 1999; 11(1):53-59. 2. Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev 1997; 77(4):1033-1079. 3. Gupta R, Sheikh A, Strachan DP, Anderson HR. Burden of allergic disease in the UK: secondary analyses of national databases. Clin Exp Allergy 2004; 34(4):520-526. 4. Kawakami T. A crucial door to the mast cell mystery knocked in. J Immunol 2009; 183(11):6861-6862. 5. Galli SJ, Nakae S, Tsai M. Mast cells in the development of adaptive immune responses. Nat Immunol 2005; 6(2):135-142. 6. Wedemeyer J, Tsai M, Galli SJ. Roles of mast cells and basophils in innate and acquired immunity. Curr Opin Immunol 2000; 12(6):624-631. 7. Gould HJ, Sutton BJ. IgE in allergy and asthma today. Nat Rev Immunol 2008; 8(3):205-217. 8. Geha RS, Jabara HH, Brodeur SR. The regulation of immunoglobulin E class-switch recombination. Nat Rev Immunol 2003;3(9):721-732. 9. Barnes PJ. Anti-IgE therapy in asthma: rationale and therapeutic potential. Int Arch Allergy Immunol 2000; 123(3):196-204. •  >\ *# ##^  =  ˜  ‚™   #  # $ ‚! $ 1999; 17:931-972. 11. Cambier JC. Antigen and Fc receptor signaling. The awesome power of the immunoreceptor tyrosine-based activation motif (ITAM). J Immunol 1995; 155(7):3281-3285. 12. Parravicini V, Gadina M, Kovarova M et al. Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat Immunol 2002; 3(8):741-748. Œ *$  `  >\  # $# # ##^  =   ‚ ~$  €€€– ’•_˜“”“• Suppl):B24-30.

MONOMERIC Ig$ % & $ $'$( $%& )*'+' ' % ^)%&+(%

43

14. Kawakami T, Galli SJ. Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2002; 2(10):773-786. 15. Furuichi K, Rivera J, Isersky C. The receptor for immunoglobulin E on rat basophilic leukemia cells: effect of ligand binding on receptor expression. Proc Natl Acad Sci USA 1985; 82(5):1522-1525. “ `$ @  #  >  =  $^ $ ##   =   $    %^ ! mast cells. Immunol Lett 1996; 52(2-3):129-134. 17. Yamaguchi M, Lantz CS, Oettgen HC et al. IgE enhances mouse mast cell Fc(epsilon)RI expression in !    !! !   !   #  =^  > ="  1997; 185(4):663-672. 18. Asai K, Kitaura J, Kawakami Y et al. Regulation of mast cell survival by IgE. Immunity 2001; 14(6):791-800. 19. Eshhar Z, Ofarim M, Waks T. Generation of hybridomas secreting murine reaginic antibodies of anti-DNP   > $ €—•– _’˜_™””^”—• 20. Tanaka S, Takasu Y, Mikura S et al. Antigen-independent induction of histamine synthesis by immunoglobulin E in mouse bone marrow-derived mast cells. J Exp Med 2002; 196(2):229-235. 21. Kitaura J, Song J, Tsai M et al. Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the FcepsilonRI. Proc Natl Acad Sci USA 2003; 100(22):12911-12916. 22. Yamada N, Matsushima H, Tagaya Y et al. Generation of a large number of connective tissue type mast cells by culture of murine fetal skin cells. J Invest Dermatol 2003; 121(6):1425-1432. _Œ ˆ< * `   *<    = ^$     $    $ in RBL-2H3 mast cells. Am J Physiol Cell Physiol 2004; 286(2):C256-263. 24. Pandey V, Mihara S, Fensome-Green A et al. Monomeric IgE stimulates NFAT translocation into the nucleus, a rise in cytosol Ca2   $    ${  # $$   # $<^_`Œ mast cell line. J Immunol 2004; 172(7):4048-4058. _ ƒ ž < > ƒ @&   =  $   #    ! #%  to those used by IgE antigen but distinct from those used by Steel factor. Blood 2003; 102(4):1405-1413. 26. Kitaura J, Kinoshita T, Matsumoto M et al. IgE- and IgE Ag-mediated mast cell migration in an autocrine/ paracrine fashion. Blood 2005; 105(8):3222-3229. 27. Kalesnikoff J, Huber M, Lam V et al. Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity 2001; 14(6):801-811. 28. Kawakami T, Kitaura J. Mast cell survival and activation by IgE in the absence of antigen: a consideration of the biologic mechanisms and relevance. J Immunol 2005; 175(7):4167-4173. 29. Kitaura J, Eto K, Kinoshita T et al. Regulation of highly cytokinergic IgE-induced mast cell adhesion by Src, Syk, Tec and protein kinase C family kinases. J Immunol 2005; 174(8):4495-4504. 30. Strober W. Immunology: The expanding T(H)2 universe. Nature 2010; 463(7280):434-435. 31. Packard KA, K Khan MM. Effects of histamine on Th1/Th2 cytokine balance. Int Immunopharmacol 2003; 3(7):909-920. 32. Goodarzi K, Goodarzi M, Tager AM et al. Leukotriene B4 and BLT1 control cytotoxic effector T-cell  $  { $ ~ $ _••Œ– ’˜•™€“^€”Œ 33. Ott VL, Cambier JC, Kappler J et al. Mast cell-dependent migration of effector CD8 T-cells through production of leukotriene B4. Nat Immunol 2003; 4(10):974-981. 34. Tager AM, Bromley SK, Medoff BD et al. Leukotriene B4 receptor BLT1 mediates early effector T-cell recruitment. Nat Immunol 2003; 4(10):982-990. 35. Makhija R, Kingsnorth AN. A Cytokine storm in acute pancreatitis. J Hepatobiliary Pancreat Surg 2002; 9(4):401-410. 36. Nakajima T, Inagaki N, Tanaka H et al. Marked increase in CC chemokine gene expression in both human and mouse mast cell transcriptomes following Fcepsilon receptor I cross-linking: an interspecies comparison. Blood 2002; 100(12):3861-3868. Œ” ƒ $  @!} ‚  \   \## Œ^<         cell function. Immunity 2002; 16(3):441-451. 38. Jolly PS, Bektas M, Olivera A et al. Transactivation of sphingosine-1-phosphate receptors by FcepsilonRI triggering is required for normal mast cell degranulation and chemotaxis. J Exp Med 2004; 199(7):959-970. 39. Matsuda K, Piliponsky AM, Iikura M et al. Monomeric IgE enhances human mast cell chemokine production: IL-4 augments and dexamethasone suppresses the response. J Allergy Clin Immunol 2005; 116(6):1357-1363. 40. Cruse G, Kaur D, Yang W et al. Activation of human lung mast cells by monomeric immunoglobulin E. Eur Respir J 2005; 25(5):858-863. 41. Gilchrest H, Cheewatrakoolpong B, Billah M et al. Human cord blood-derived mast cells synthesize and release I-309 in response to IgE. Life Sci 2003; 73(20):2571-2581.

44

MAST CELL BIOLOGY

42. Borkowski TA, Jouvin MH, Lin SY et al. Minimal requirements for IgE-mediated regulation of surface Fc epsilon RI. J Immunol 2001; 167(3):1290-1296. 43. K Kubo S, Matsuoka K, Taya C et al. Drastic up-regulation of Fcepsilonri on mast cells is induced by IgE binding through stabilization and accumulation of Fcepsilonri on the cell surface. J Immunol 2001; 167(6):3427-3434. 44. Kitaura J, Xiao W, Maeda-Yamamoto M et al. Early divergence of Fc epsilon receptor I signals for receptor up-regulation and internalization from degranulation, cytokine production and survival. J Immunol 2004; 173(7):4317-4323. ’ >  * ` ‚#$# ‚   ^%  "      #$   $  =  ‚^  !  " % <   !!  {  responses. BMC Genomics 2006; 7:210. 46. Yip KH, Huang Y, Waye MM et al. Induction of nitric oxide synthases in primary human cultured mast   =   {  <  $#  _••—– —˜™”“’^”“— 47. Nevin BJ, Broadley KJ. Nitric oxide in respiratory diseases. Pharmacol Ther 2002; 95(3):259-293. 48. Fischer A, Folkerts G, Geppetti P et al. Mediators of asthma: nitric oxide. Pulm Pharmacol Ther 2002; 15(2):73-81. 49. Watanabe M, Satoh T, Yamamoto Y et al. Overproduction of IgE induces macrophage-derived chemokine (CCL22) secretion from basophils. J Immunol 2008; 181(8):5653-5659. 50. Saffar AS, Alphonse MP, Shan L et al. IgE modulates neutrophil survival in asthma: role of mitochondrial pathway. J Immunol 2007; 178(4):2535-2541. 51. Rowland SL, Depersis CL, Torres RM et al. Ras activation of Erk restores impaired tonic BCR R signaling and rescues immature B-cell differentiation. J Exp Med 2010. 52. Kawakami Y, Kitaura J, Yao L et al. A Ras activation pathway dependent on Syk phosphorylation of protein kinase C. Proc Natl Acad Sci USA 2003; 100(16):9470-9475. 53. Blank U, Cyprien B, Martin-Verdeaux S et al. SNAREs and associated regulators in the control of exocytosis in the RBL-2H3 mast cell line. Mol Immunol 2002; 38(16-18):1341-1345. 54. Puri N, Roche PA. Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis. *   _••“– ”˜™’—_^’€’ 55. Kashiwakura J, Xiao W, Kitaura J et al. Pivotal advance: IgE accelerates in vitro development of mast cells    # # > ƒ$< † _••—– —’˜_™Œ”^Œ“” “ ƒ$ * †# >&   =ƒ     #^  $  =     isolation and characterization. J Immunol 1980; 124(6):2728-2737. 57. MacDonald SM, Lichtenstein LM, Proud D et al. Studies of IgE-dependent histamine releasing factors: heterogeneity of IgE. J Immunol 1987; 139(2):506-512. 58. Schroeder JT, Lichtenstein LM, MacDonald SM. An immunoglobulin E-dependent recombinant histamine-releasing factor induces interleukin-4 secretion from human basophils. J Exp Med 1996; 183(3):1265-1270. 59. Gould HJ, Sutton BJ, Beavil AJ et al. The biology of IGE and the basis of allergic disease. Annu Rev Immunol 2003; 21:579-628. 60. Beaven MA, Metzger H. Signal transduction by Fc receptors: the Fc epsilon RI case. Immunol Today 1993; 14(5):222-226. 61. Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell 1990; 61(2):203-212. 62. Heldin CH. Dimerization of cell surface receptors in signal transduction. Cell 1995; 80(2):213-223. 63. Field KA, Holowka D, Baird B. Fc epsilon RI-mediated recruitment of p53/56lyn to detergent-resistant membrane domains accompanies cellular signaling. Proc Natl Acad Sci USA 1995; 92(20):9201-9205. “’   `%<  †  † @ } !  # ##   $$ E receptor within membrane domains. J Biol Chem 1997; 272(7):4276-4280. “ > ƒ@ ‚!  \ *% <   $      !   2003; 299(5611):1362-1367. 66. Schramm G, Mohrs K, Wodrich M et al. Cutting edge: IPSE/alpha-1, a glycoprotein from Schistosoma mansoni eggs, induces IgE-dependent, antigen-independent IL-4 production by murine basophils in vivo. J Immunol 2007; 178(10):6023-6027. 67. Charles N, Monteiro RC, Benhamou M. p28, a novel IgE receptor-associated protein, is a sensor of receptor occupation by its ligand in mast cells. J Biol Chem 2004; 279(13):12312-12318. 68. Jayawardana ST, Ushio H, Niyonsaba F et al. Monomeric IgE and lipopolysaccharide synergistically prevent mast-cell apoptosis. Biochem Biophys Res Commun 2008; 365(1):137-142. 69. Kohno M, Yamasaki S, Tybulewicz VL et al. Rapid and large amount of autocrine IL-3 production is responsible for mast cell survival by IgE in the absence of antigen. Blood 2005; 105(5):2059-2065. 70. Shelburne CP, McCoy ME, Piekorz R et al. Stat5 expression is critical for mast cell development and survival. Blood 2003; 102(4):1290-1297.

MONOMERIC Ig$ % & $ $'$( $%& )*'+' ' % ^)%&+(%

45

71. Sly LM, Kalesnikoff J, Lam V et al. IgE-induced mast cell survival requires the prolonged generation of reactive oxygen species. J Immunol 2008; 181(6):3850-3860. 72. Yanagida M, Fukamachi H, Ohgami K et al. Effects of T-helper 2-type cytokines, interleukin-3 (IL-3), IL-4, IL-5 and IL-6 on the survival of cultured human mast cells. Blood 1995; 86(10):3705-3714. 73. Oskeritzian CA, Zhao W, Pozez AL et al. Neutralizing endogenous IL-6 renders mast cells of the MCT type from lung, but not the MCTC type from skin and lung, susceptible to human recombinant IL-4-induced apoptosis. J Immunol 2004; 172(1):593-600. ”’ *<< ` '# ` ~      $  {  <  $ from murine mast cells by monomeric IgE and toll-like receptor ligands. Biochem Biophys Res Commun 2010; 391(1):471-476. 75. Yoshikawa H, Nakajima Y, Tasaka K. Glucocorticoid suppresses autocrine survival of mast cells by inhibiting IL-4 production and ICAM-1 expression. J Immunol 1999; 162(10):6162-6170. 76. Yoshikawa H, Nakajima Y, Tasaka K. Enhanced expression of Fas-associated death domain-like IL-1-converting enzyme (FLICE)-inhibitory protein induces resistance to Fas-mediated apoptosis in activated mast cells. J Immunol 2000; 165(11):6262-6269. 77. Alfredsson J, Puthalakath H, Martin H et al. Proapoptotic Bcl-2 family member Bim is involved in the control of mast cell survival and is induced together with Bcl-XL upon IgE-receptor activation. Cell Death Differ 2005; 12(2):136-144. ”— ‚!  >     $ $    ! >   @ $ _••“– 117(6):1214-1225 79. Malbec O, Malissen M, Isnardi I et al. Linker for activation of T-cells integrates positive and negative signaling in mast cells. J Immunol 2004; 173(8):5086-5094. 80. Nunomura S, Gon Y, Yoshimaru T et al. Role of the FcepsilonRI beta-chain ITAM as a signal regulator for mast cell activation with monomeric IgE. Int Immunol 2005; 17(6):685-694. 81. Kawakami Y, Kitaura J, Hartman SE et al. Regulation of protein kinase CbetaI by two protein-tyrosine kinases, Btk and Syk. Proc Natl Acad Sci USA 2000; 97(13):7423-7428. 82. Liu Y, Furuta K, Teshima R et al. Critical role of protein kinase C betaII in activation of mast cells by monomeric IgE. J Biol Chem 2005; 280(47):38976-38981. 83. Yamasaki S, Saito T. Progress in allergy signal research on mast cells: signal regulation of multiple mast cell responses through FcepsilonRI. J Pharmacol Sci 2008; 106(3):336-340. 84. Sakurai D, Yamasaki S, Arase K et al. Fc epsilon RI gamma-ITAM is differentially required for mast cell function in vivo. J Immunol 2004; 172(4):2374-2381. 85. Yamasaki S, Ishikawa E, Kohno M et al. The quantity and duration of FcRgamma signals determine mast cell degranulation and survival. Blood 2004; 103(8):3093-3101. —“  # @>       <    ! $ $ " $ signal-regulated kinase activation. Cell 1995; 80(2):179-185. 87. Werlen G, Hausmann B, Naeher D et al. Signaling life and death in the thymus: timing is everything. Science 2003; 299(5614):1859-1863. 88. Yamasaki S, Ishikawa E, Sakuma M et al. LAT and NTAL mediate immunoglobulin E-induced sustained extracellular signal-regulated kinase activation critical for mast cell survival. Mol Cell Biol 2007; 27(12):4406-4415. 89. Yamasaki S, Takase-Utsugi M, Ishikawa E et al. Selective impairment of FcepsilonRI-mediated allergic   ^    $ _••—– _•˜•™_—€^_€” 90. Tanaka S, Mikura S, Hashimoto E et al. Ca2 {$"^ # #  ƒ^“    cells activated by monomeric IgE. Eur J Immunol 2005; 35(2):460-468. € $<  * @ $    =#  ^  =     $ # and immunobiological roles of IgE in vivo. Int Immunol 1999; 11(6):987-994. €_ ˆ `@   *‚ &#%^†     ! #"  =^   ~$  €€’– 370(6488):367-370. 93. Gurish MF, Bryce PJ, Tao H et al. IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis. J Immunol 2004; 172(2):1139-1145. 94. Lantz CS, Boesiger J, Song CH et al. Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites. Nature 1998; 392(6671):90-93. 95. Matsuda H, Watanabe N, Geba GP et al. Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice. Int Immunol 1997; 9(3):461-466. 96. Pritchard DI, Hewitt C, Moqbel R. The relationship between immunological responsiveness controlled by T-helper 2 lymphocytes and infections with parasitic helminths. Parasitology 1997; 115 Suppl:S33-44. 97. Bryce PJ, Miller ML, Miyajima I et al. Immune sensitization in the skin is enhanced by antigen-independent effects of IgE. Immunity 2004; 20(4):381-392. €— # @†  # => @ †   = {$ # $  $  $    $     $    $  { > $ _••€– —_˜’™_’“^_’_’

46

MAST CELL BIOLOGY

99. Kitamura Y, Ito A. Mast cell-committed progenitors. Proc Natl Acad Sci USA 2005; 102(32):11129-11130. 100. Kitamura Y, Shimada M, Hatanaka K et al. Development of mast cells from grafted bone marrow cells in irradiated mice. Nature 1977; 268(5619):442-443. 101. Nabel G, Galli SJ, Dvorak AM et al. Inducer T-lymphocytes synthesize a factor that stimulates proliferation of cloned mast cells. Nature 1981; 291(5813):332-334. 102. Galli SJ, Zsebo KM, Geissler EN. The kit ligand, stem cell factor. Adv Immunol 1994; 55:1-96. 103. Okayama Y, Kawakami T. Development, migration and survival of mast cells. Immunol Res 2006; 34(2):97-115. •’ †  =ƒ @ <  * # $   $  #  $    #$ Microbiol Rev 1987; 51(2):192-205. 105. Bienenstock J, Befus AD, Pearce F et al. Mast cell heterogeneity: derivation and function, with emphasis on the intestine. J Allergy Clin Immunol 1982; 70(6):407-412. 106. McNeil HP, Austen KF, Somerville LL et al. Molecular cloning of the mouse mast cell protease-5 gene. A novel secretory granule protease expressed early in the differentiation of serosal mast cells. J Biol Chem 1991; 266(30):20316-20322. 107. Reynolds DS, Gurley DS, Austen KF et al. Cloning of the cDNA and gene of mouse mast cell protease-6. Transcription by progenitor mast cells and mast cells of the connective tissue subclass. J Biol Chem 1991; 266(6):3847-3853. 108. Reynolds DS, Stevens RL, Gurley DS et al. Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family. J Biol Chem 1989; 264(33):20094-20099. •€    &= ‚  ‚‘       $    ! $ $ mast cell serine protease. J Biol Chem 1990; 265(1):423-429. •    &= $! *\ @    @  # ~    $     ’           $    #  $^  neutral proteases of the mouse and rat. J Biol Chem 1991; 266(3):1934-1941. 111. Trong HL, Newlands GF, Miller HR R et al. Amino acid sequence of a mouse mucosal mast cell protease. Biochemistry 1989; 28(1):391-395. 112. Wright HV, Bailey D, Kashyap M et al. IL-3-mediated TNF production is necessary for mast cell development. J Immunol 2006; 176(4):2114-2121. 113. Kawakami Y, Yumoto K, Kawakami T. An improved mouse model of atopic dermatitis and suppression of skin lesions by an inhibitor of Tec family kinases. Allergol Int 2007; 56(4):403-409. 114. Kawakami Y, Tomimori Y, Yumoto K et al. Inhibition of NK K cell activity by IL-17 allows vaccinia virus to induce severe skin lesions in a mouse model of eczema vaccinatum. J Exp Med 2009; 206(6):1219-1225. 115. Kashiwakura J, Kawakami Y, Yuki K et al. Polyclonal IgE induces mast cell survival and cytokine production. Allergol Int 2009; 58(3):411-419.

CHAPTER R4 MAST CELL APOPTOSIS AND SURVIVAL

  =<  $ ~| Clinical Immunology and Allergy, Department of Medicine, Karolinska Institutet, Stockholm, Sweden      %     "

Abstract:

Mast cells are enigmatic cells that are recognized as critical components of our immune system. They are strategically localized at the host/environment interface, display a long lifespan once situated in tissues and have the ability to produce, store  $ !  $^ $  $    $  chronic conditions, mast cell accumulation, activation and release of mediators are    #    $  # {  %# these disease states. During the past decade, regulatory pathways for mast cell survival have been elucidated, which have, in part, helped to explain the increased $  $ !!     ! $  {    with, e.g., the allergic response. One key group of regulators involved in cell survival and apoptosis is the Bcl-2 family of proteins. The Bcl-2 family consists of pro- and anti-apoptotic members, where the balance between these members determines cellular fate via protein-protein interactions. In this chapter, we will discuss the regulation of mast cell apoptosis and survival and how understanding the mechanisms by which Bcl-2 family members regulate mast cell survival could   #    <   #  # !   { This knowledge could be used to develop treatments for mast cell disorders such    # {   %#      !!

INTRODUCTION Mast cells have primarily been regarded as effector cells in allergic disorders,   ^{    $ =^ ¡RI crosslinking. However, their strategic distribution and their capacity to secrete potent factors, indicate that they can take part in various immune responses. There has been a recent renaissance in mast cell research which has resulted in an appreciation of their other Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 47

48

MAST CELL BIOLOGY

roles beyond atopic diseases.1 In this respect mast cells are now recognized to play a key role in both host defence and in many different diseases, where they either can be protective or promote the disease. An accumulation of mast cells is often observed at sites of both bacterial and viral infections, or in parasitic diseases where the release of mast cell mediators contributes to an immune response (reviewed in ref. 2). During viral infections, mast cells release mediators distinct from those induced by a bacterial infection and these mediators then selectively recruit T cells and natural killer cells, rather than neutrophils, to the site of infection.3 In response to nematodes and other parasites, mast cells and in particular mast cell protease (MCP)-14 is critical for parasite expulsion. Mast cells have also been associated with autoimmune diseases, such as multiple sclerosis (MS) and rheumatoid arthritis (RA). The most compelling evidence for the involvement of mast cells in MS comes from the use of an animal model of experimental allergic encephalomyelitis (EAE) where mice lacking mast cells develop less severe EAE and at a later onset.5 In RA, mast  #!  $   $  # #  {   $  joints.6 `%!  #   #  ^ ! ƒ^• $  $<    {  $    $     7 cautions us not to see mast cells as simply detrimental for a disease. Mast cell numbers have been found to increase within the airway smooth muscle of asthmatic patients8 and the current view is that mast cells contribute to the immediate reaction and to the progression of the later chronic reaction (reviewed in ref. 9). Mast cells are also often found in association with many different tumours; and studies indicate that mast cells are associated with angiogenesis in some tumours and can promote growth and metastasis via the release of various mediators.10-12 *#  imply that mast cells might participate in tumorogenesis rather than providing a defence against these tumours. Since mast cells appear to be involved in a variety of diseases, the possibility to use them as direct targets for new therapeutical interventions is attractive. A potential way of doing this is through the regulation of mast cell numbers and their activity within $ #   #   $  #$ # !   {

MAST CELL BIOLOGY Mast cells are found in tissues throughout the body but originate from haematopoietic stem cells in the bone marrow.13,14 Unlike many other haematopoietic cells that leave the bone marrow fully matured, mast cells stem from immature precursors that leave the bone marrow to circulate in the blood. Once the cells are recruited into the peripheral $ #  $ # {$     ˜@™  #   $ cytokines, differentiate into mature mast cells.15 The surrounding microenvironment in $    $   %  #          # # # { #  $ #   $ 16 Mature mast cells do not represent a homogenous population and are usually divided  %  $ ‚        ! $  cells (CTMC) and mucosal mast cells (MMC).17 MMCs depend, in addition to SCF, on T-cell-derived cytokines, whereas CTMCs primarily require SCF for their persistence.

    *  < #

@ $ %#  # @* @ $ #  *^ dependence and subsequently appear at normal numbers.18 `$      

MAST CELL APOPTOSIS AND SURVIVA V L

49

based on the expression of proteases in their granules, into mast cells containing tryptase (MCT) and mast cells containing tryptase and chymase (MCTC).19 In terms of tissue localization, human MCT correspond to rodent MMC, being located predominantly in mucosal tissues, whereas human MCTC correspond to rodent CTMC cells and are mainly found in connective tissues.20 *#    %# #  # #$ %#  *^ $  < @T in the intraepithelial compartment of their intestine, while MCTC appear at normal numbers in the surrounding connective tissues.21 Mast cells normally survive within tissues for several months and their numbers are kept relatively constant. However, intestinal helmith infection of rodents will generate a TH_^ {   $ 

@ #  %##   for the expulsion of the worm.22 Mast cells arising during the TH2 immune response undergo dramatic changes in their granule architecture; and their content of proteases and proteoglycans, due to the milieu of T-cell-derived cytokines.22-24   # {$ of TH2-derived cytokines can be seen in allergic diseases of humans, where they are #$#   $  #    #          cell numbers in the mucosal epithelium of the nose, bronchi and gastrointestinal tracts,25 linking TH_^ {   %#   # 26    $ {   $$   #  # {     $     !  {   $#    asthma, various skin diseases, autoimmune disease like rheumatoid arthritis and tumours. In several of these diseases, a correlation between mast cell numbers and disease severity has been described.27-31 Therefore, it would be desirable to be able to regulate mast cell numbers and their activity within the tissue, with the hypothesis that a reduction of mast cell numbers would generate lesser symptoms.

REGULATION OF APOPTOSIS AND SURVIVAL Apoptosis, or programmed cell death, is vital for the development and health of a multicellular organism. Apoptosis is used during the development of an organism to sculpture organs and tissues and to ensure immunity. Later in life, apoptosis protects by executing deletion of damaged, aged or potentially dangerous cells. Thereby homoeostasis, the balance between cell proliferation and death, is maintained. During apoptosis, cells die in a controlled, regulated way in response to various stimuli such as growth factor deprivation, DNA damage, developmental signals, cellular stress and cytotoxic drugs. The sensitivity of cells to apoptotic signals can vary depending on a number of factors such as the nature of the stimuli, the stage of the cell cycle and the expression of pro- and anti-apoptotic proteins. Dysregulation of apoptosis can lead to #  "       #    %#  !   32 In cancer and autoimmune diseases, cells commonly escape apoptosis by overexpressing anti-apoptotic proteins33,34 thus making inhibition of these proteins an attractive strategy for therapy. Apoptosis is regulated via two parallel but converging pathways, the extrinsic and the intrinsic pathway (Fig. 1). Both pathways commonly involve caspase activation and lead to targeted degradation of cellular structures and formation of apoptotic bodies.35 The extrinsic pathway is stimulated via external signals from the environment outside the cell.36 The signal is transmitted through death receptors, belonging to the TNF receptor family (such as Fas/CD95R and the receptor for Tumor necrosis (TNF)-related

50

MAST CELL BIOLOGY

Figure 1. The extrinsic and intrinsic pathway of apoptosis. Left: The extrinsic pathway-induced apoptosis is triggered by ligation of death receptors (such as Fas or TRAIL). Death receptor stimulation-mediated   ! ^—   %##   ^Œ !    # Upon death receptor stimulation activated caspase-8 cleaves Bid into truncated Bid (tBid) that can interact with the intrinsic pathway. Right: The intrinsic pathway responds to stress factors such as DNA damage, UV radiation and cytokine deprivation that initiate apoptosis by activating the BH3-only proteins. These can neutralize the anti-apoptotic proteins which lead to activation of Bax and Bak. Some of the BH3-only proteins may also be able to directly activate Bax and Bak. Once activated, Bax and Bak cause outer mitochondrial disruption and release of cytochrome c that together with caspase-9, Apaff 1and ATP form an apoptosome that trigger the activation of caspase-9 and downstream effector caspases like caspase-3 that execute cell death.

apoptosis-inducing ligand (TRAIL-R), located on the surface of the cell; and which lead to the activation of the caspase cascade via caspase-8 (caspase-10 in humans). The intrinsic or mitochondrial pathway responds to stress factors such as growth factor deprivation, DNA damage and other stimuli that originate from the inside of the cell.37,38 The Bcl-2 family of proteins plays a key role in the intrinsic pathway of apoptosis. Their primary site of action is at the mitochondrial membrane where the relative balance of Bcl-2 family members determines if mitochondrial outer membrane permeabilisation (MOMP) will occur. Following MOMP, apoptogenic factors, such as cytochrome c, will be released. This leads to the activation of Apoptotic protease activating factor 1 (Apaff 1) and the formation of the apoptosome; consisting of cytochrome c, Apaff1 and adenosine triphosphate (ATP). The apoptosome activates caspase-9 which in turn induces the activation of effector caspases that cause proteolytic degradation and subsequent cell death.39

MAST CELL APOPTOSIS AND SURVIVA V L

51

Since caspases are effectors of both the extrinsic and intrinsic pathways, their activities need to be strictly regulated. The Inhibitor of Apoptosis Proteins (IAP) family interacts with caspases and as a consequence inhibits their proteolytic activity.40 IAP members in turn are regulated by the Smac/DIABLO protein which is released from mitochondria during the apoptotic process.41 Although the two pathways are independent from each other42 crosstalk does occur. This is mediated via Bid, a member of the Bcl-2 family, which can be activated by caspase-8 (or caspase-10 in humans)-mediated proteolysis which generates two fragments. The truncated Bid fragment (tBid) has the capacity to {$ #   #%43 The Bcl-2 protein family contains members that safeguard cell survival (anti-apoptotic proteins) as well as proteins that promote apoptosis (pro-apoptotic  ™      #  }   #!    *# †^_  members are characterized by the presence of at least one and up to four Bcl-2 homology ˜†`™  *# ^   ˜$ †^_ †^ƒ †^% †{^ and Mcl-1) contain up to four BH domains (BH1-4) and a trans-membrane region (TM) that allows targeting to intracellular membranes. The pro-apoptotic members can be sub-divided into two groups: BH3 domain only proteins (BH3-only; such as Bcl-XS, Bad, Bim, Bmf, Bid, Puma and Noxa), which share with each other only the BH3 domain; and Bax/Bak-like proteins (Bax, Bak and Bok; a non-extensively studied Bax/Bak-like protein), which contain BH1, BH2 and BH3 domains and a trans-membrane region.39,44 Regulation of apoptosis is dependent on the balance between the different Bcl-2 family members and through the interactions between the proteins within the three subgroups. Individual BH3-only proteins differ in their ability to bind to anti-apoptotic †^_     #   !  # $  # $45,46 (Fig. 2). The Bim, Puma and tBid proteins, being more promiscuous in their binding

Figure 2. BH3-only proteins differ in their ability to bind to anti-apoptotic Bcl-2 family members. Bim, Bid and Puma binds promiscuously to all anti-apoptotic proteins while Bad only binds to Bcl-2, Bcl-XL and Bcl-w and Noxa to A1 and Mcl-1.

52

MAST CELL BIOLOGY

to anti-apoptotic members, have the capacity to induce apoptosis on their own, while the more selective Noxa and Bad must cooperate in order to do this. Following the interaction of BH3-only proteins and anti-apoptotic Bcl-2 proteins, which targets the cell for apoptosis, execution requires activation of Bax/Bak-like proteins.47,48 Although it is well-known that Bcl-2 family members do interact, different modes of action have been proposed for the interactions between anti-apoptotic and pro-apoptotic Bcl-2 family proteins (reviewed in ref. 29). Targeting the Bcl-2 family is one of the strategies for the development of therapeutic treatments against diseases associated with dysregulation or dysfunction of apoptosis. A way of targeting the Bcl-2 family has been the development of drugs that mimic the binding of BH3-only proteins to the anti-apoptotic proteins, thus inhibiting their function and promoting apoptosis. This has already proven useful in cancer therapy where the use of BH3 mimetics sensitizes tumour cells to apoptosis.50

MAST CELL APOPTOSIS Mature mast cells situated in tissues are long-lived and primarily depend on SCF for their survival. The importance of SCF is highlighted by the almost complete lack of          @51-53 and reducing SCF production reduces mast cell numbers in tissues.54 In vitro-derived mast cells deprived of their requisite growth factors and thereby destined for apoptosis can be rescued by the addition of SCF.55,56 $ #   #   $ #     < #  induces mast cell apoptosis in the synovia.57 It has been shown that SCF regulates mast cell survival through inactivation of FOXO3a and down-regulation and phosphorylation of its target, the pro-apoptotic BH3-only protein, Bim58 subsequently leading to the ubiquitination and proteasomal degradation of Bim.59-61  !  †^   cells are also found to be partially resistant to cytokine deprivation-induced apoptosis in culture.62 Since overexpression of Bcl-2 protected mast cells more potently than loss of Bim, other pro-apoptotic BH3-only proteins, besides Bim, are likely to be involved in this process. A way to unravel the role of Bcl-2 family members in the regulation of mast cell $ !!    # $  †^_  ^     *# role of other BH3-only proteins, besides Bim, in the regulation of mast cell survival   #   $ *# #   #    # BH3-only protein Puma as critical for the induction of mast cell apoptosis following cytokine deprivation. Upon cytokine deprivation-induced apoptosis, both Bim and Puma are upregulated with resulting induction of apoptosis in the mast cells58,63 and mast cells lacking either Bim or Puma show an increased viability upon cytokine deprivation compared to wild type (wt) mast cells. The combined loss of both Bim and Puma offers the same level of protection as overexpression of Bcl-2, demonstrating the importance of a tight regulation of Bim and Puma for mast cell survival, since both bind with high     ^ †^_    # #  The importance of the effector proteins Bax and Bak is demonstrated by the fact that their absence abolishes most apoptotic responses sensed by BH3-only proteins.47 Loss of both Bak and Bax results in embryonic lethality,64 %#     in Bax or Bak will develop more or less normally.64,65 Although both Bax and Bak

MAST CELL APOPTOSIS AND SURVIVA V L

53

have been shown to have largely overlapping functions in many cells, it has also been reported that one of them can have a dominant role over the other in certain cell types.64,66 Increased mast cell numbers are found in the stomach mucosa of mice lacking Bax or Bak; and in the back skin of mice lacking Bak.67 This may indicate a more prominent role for Bax in MMCs compared to CTMCs for cell homeostasis. Furthermore, loss of Bax conferred protection from cytokine-deprivation induced apoptosis of in vitro-derived BMMCs.67 A recent study demonstrates both Bax and Bak are present in in vitro-derived mast cells, although the levels of Bax appear to exceed that of Bak.68 ' < %# % †<^           %   %#  †"^         for both Bak and Bax were partially or completely resistant to apoptosis, respectively. Notably, only combined loss of both bax and bakk confers complete resistance to cytokine deprivation-induced apoptosis, which demonstrates that activated Bak does play a role in mast cell apoptosis, though to a lesser extent than activated Bax. The          # †<  †" %         #  ^ †  \$    ! "  anti-apoptotic Bcl-2. Downstream of the Bax and Bak proteins, Apaff 1 and caspase-9 carry out their functions in the intrinsic pathway of apoptosis. It has been shown that lymphocytes lacking either of these proteins still have the capacity to undergo apoptosis in response to growth factor deprivation or DNA damage.69 In contrast, mast     # ^    ^€   $      cytokine deprivation and instead remain in a ‘senescent’ stage with no proliferative capability and with lost effector functions.70 Many Bcl-2 family member gene deletions in mice lead to disrupted homeostasis and, as a consequence, cells accumulate in excess. This might cause increased tumour incidence and autoimmunity in mice reaching adulthood.71,72 The lack of certain Bcl-2 family members can have implications during the development of mast cells both in !    !!     ^  % !    # and display mast cell numbers in the tissues comparable to wild-type mice.73,74 In   ^ †^_ †^ƒ ^  †^%      $  development in mice.75,76 In the absence of the genes bcl-x or bcl-2, no offspring are born or are born with a short life expectancy, respectively.76,77 It is, however, possible to generate mast cells from murine embryonic stem cells lacking bcl-x or bcl-2, although # ‹$  # @  ƒ^Œ   $   %     to wild type embryonic stem cells.78 The importance of the anti-apoptotic Bcl-2 protein for mast cell survival is also highlighted by the fact that inhibition of Bcl-2, using single chained antibodies against Bcl-2, induces mast cell apoptosis. The protein-protein interaction of Bcl-2 is then   $ }  $  #  #  ^ $79 Since mast cells can undergo apoptosis via the intrinsic pathway, it seems likely that mast cells might also undergo apoptosis in response to activation through death receptors/the extrinsic pathway, thus providing an additional pathway that could contribute to the regulation of mast cell numbers. The expression of death receptors @€‚  *‚ƒ^‚ #!     # $   #$   although induction of apoptosis through these receptors varies. Fas/CD95R R activation has been found to induce apoptosis in murine mast cells,80 whereas TRAIL-R crosslinking causes an increase in human mast cell apoptosis.81

54

MAST CELL BIOLOGY

MAST CELL SURVIVAL Mast cells have the ability to survive the aggregation of Fc¡RI (IgE/Fc¡RI crosslinking) and the subsequent degranulation process which is a key event in perpetuating # {  82,83 This increased survival of both human and murine mast cells (activation-induced survival) following IgE/Fc¡RI crosslinking has been demonstrated.74,84-86 A fundamental question in mast cell biology is how this survival is mediated. Opposing results have however, been reported regarding the effects of enhanced mast cell survival following IgE/Fc¡RI crosslinking.87,88 These differences may be due to IgE type, strength and duration of the crosslinking, or the mast cell phenotype.85,89-91 One study showed that weak to moderate Fc¡RI crosslinking led to activation-induced survival, while stronger Fc¡RI crosslinking resulted in increased degranulation and IL-6 production but diminished survival.85 Moreover, with the use of a CD8 fusion protein, the strength of Fc¡RI signals was manipulated, demonstrating activation-induced survival even in the absence of degranulation.90 † # $   # $  !!  $ # $ {$ the mast cell response. There is a local accumulation of IgE bearing MCTs upon Ag exposure in seasonal allergic rhinitis, whereas MCTCs do not increase.92 In vitro, mast cell populations have been shown to differ in their ability to survive allergic reactions, where the phenotype of the mast cells seems to determine if the cells are to induce a survival program upon allergic activation or not. While cytokine-deprived mast cells of connective tissue-like subtype exhibit Fc¡RI stimulation-induced upregulation of anti-apoptotic A1 and enhanced cell survival in vitro, mast cells of the mucosal-like type do not.91 U Upon Fc¡RI crosslinking, mouse bone marrow derived mast cells (BMMCs) upregulate anti-apoptotic Bcl-2 family member A174 and Bcl-XL and also, to a lesser extent, Bcl-2 on an mRN R A level.62,93  #   #% # $         exhibit activation-induced survival upon Fc¡RI crosslinking.74   ^  mice display mast cell numbers in the tissue comparable to wild type (wt) mice.74 However, $ }  $‹$  ! %#   ^   #%  lower number of mast cell compared to wt mice.74 The human homologue of A1, &'*+, is upregulated in human mast cells upon Fc¡RI aggregation94 and it was also shown that human cord blood derived mast cells (CBMCs), following Fc¡RI crosslinking, upregulate the anti-apoptotic Bcl-2 family member Mcl-1.88,94 These observations provide an explanation for IgE-mediated activation-induced mast cell survival, demonstrating a ‘     $     $ # # #$ #$ †{^ $ be of importance in human mast cells for this process. Interestingly, Fc¡RI crosslinking not only regulates anti-apoptotic proteins of the Bcl-2 family, but also pro-apoptotic proteins are affected. In murine mast cells, Fc¡RI crosslinking upregulates and induces a rapid, but transient, phosphorylation of Bim.62 Other BH3-only proteins including Bad are also phosphorylated, thereby inhibiting their pro-apoptotic functions.95 In CBMCs, Fc¡RI crosslinking increases TRAIL-induced caspase-8 and caspase-3 activation; and the levels of FLICE-like inhibitory protein (FLIP), Mcl-1 and Bim are upregulated while Bid is downregulated.88 The conundrum of Bim upregulation following Fc¡RI crosslinking needs further investigation. It has been described that the interaction between A1 and Bim increases the half life of A1 and    ^ 96 and it is possible that the transient phosphorylation of Bim inhibits its apoptotic effect.62

MAST CELL APOPTOSIS AND SURVIVA V L

55

Figure 3. The balance and interactions between different anti-apoptotic and pro-apoptotic activities/ proteins determine whether the mast cell will survive or undergo apoptosis upon IgE/Fc¡RI crosslinking.

Given that pro- and anti-apoptotic Bcl-2 family proteins can bind to each other and titrate each others function suggests that the regulation of the aforementioned Bcl-2 family members, both anti-apoptotic as well as pro-apoptotic, following Fc¡RI crosslinking is likely to determine cell fate (Fig. 3).

MASTOCYTOSIS Mastocytosis is a disorder characterized by an abnormal accumulation of malignant mast cells in one or several organs.97 In systemic mastocytosis, most of the neoplastic mast cells have an activating mutation in c-kit, the receptor for SCF, causing an auto-activation of the receptor.97-100 In mast cells, SCF normally regulates survival by repressing the levels of pro-apoptotic BH3-only protein Bim.58 SCF affects both the transcription of Bim by inactivating its transcription factor and by phosphorylation which leads to proteasomal degradation of the protein. Recent data suggests Bim is expressed at low levels in neoplastic mast cells in systemic mastocytosis; and that drug-induced re-expression of Bim is associated with inhibition of proliferation and diminished survival.101 Furthermore, neoplastic mast cells express the anti-apoptotic Bcl-2 family protein Mcl-1 constitutively; and targeting Mcl-1 in these cells is associated with reduced growth and induction of apoptosis.102 This also suggests the anti-apoptotic Bcl-2 family member Mcl-1 is a novel, interesting target in treating mastocytosis. An overexpression and dysregulation of pro-survival Bcl-2 and Bcl-XL has also been detected in mastocytosis and may also contribute to the pathogenesis of the disease.103-105 Thus, small molecular inhibitors of crucial Bcl-2 family members involved in mast cell survival and/or apoptosis offer an attractive strategy for the treatment of this disease.

CONCLUSION

  #!      #    $  {    %#    # ‚$ # !   # {

56

MAST CELL BIOLOGY

by decreasing mast cell numbers, for example through induction of apoptosis, has been suggested as an alternative therapeutic treatment in mast cell-associated diseases. In order to achieve this objective, we need to understand how mast cell survival and apoptosis is regulated in these settings. Pro- and anti-apoptotic proteins belonging to the Bcl-2 family are known to regulate apoptosis and, although our knowledge of how these regulate mast cell survival and apoptosis is advancing, the structural and functional differences between the members and their relative impact on mast cell survival and apoptosis need        <% #%!  # ^ †^_       neutralize, pro-apoptotic members. This suggests that, once the key Bcl-2 family members #  # !   { #!    # $   $ inhibitors could block their binding and thereby sensitize mast cells for apoptosis. A way of targeting the Bcl-2 family has been the development of drugs that mimic the binding of BH3-only proteins to the anti-apoptotic proteins thereby inhibiting their function and thus promoting apoptosis. This has already proven useful in cancer therapy where the use of BH3 mimetics sensitizes tumour cells to apoptosis.50 ABT-737 is a small molecular inhibitor, mimicking the binding BH3 domain of the pro-apoptotic protein †   %# ##    # ^   †^ƒ †^_  †^% but not Mcl-1 or A1.106  #       $  #    solid tumors and lymphoid malignancies, although overexpression of Mcl-1 can make cells refractory to the drug.107-111 In allergy, a way to sensitise mast cells for apoptosis would be to inhibit the interactions   ^$ !! †{^ %#    $      In allergy a way to sensitise mast cells for apoptosis would be to inhibit the interactions   ^$ !! †{^ %#           $ #   $  # ^ $  †{^     %# # †^_    #!       %#  †{^ % overexpressed. Hopefully the recent derived crystal structures of both A1112  †{^113 %   $ $   #% †{^ "   ^ $   when designing a small molecular inhibitor to abrogate the interactions of anti-apoptotic †{^ %#   $      Although systemic abolishment of mast cells is not desirable, targeting of mast cells %#    $ $ #!   # $  ~%       $ !! #$   # !   {   ^ disorders.

REFERENCES 1. Leslie M. Mast cells show their might. Science 2007; 317:614-616. 2. Marshall JS. Mast-cell responses to pathogens. Nat Rev Immunol 2004; 4:787-799. 3. Dawicki W, Marshall JS. New and emerging roles for mast cells in host defence. Curr Opin Immunol 2007; 19:31-38. 4. K Knight PA, Wright SH, Lawrence CE et al. Delayed expulsion of the nematode Trichinella spiralis in mice < # $  ^   $ # $    ^ > ="  _•••– 192:1849-1856. 5. Secor VH, Secor WE, Gutekunst CA et al. Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis. J Exp Med 2000; 191:813-822. 6. Maruotti N, Crivellato E, Cantatore FP et al. Mast cells in rheumatoid arthritis. Clin R Rheumatol 2007; 26:1-4. 7. Grimbaldeston MA, Nakae S, Kalesnikoff J et al. Mast cell-derived interleukin 10 limits skin pathology in contact dermatitis and chronic irradiation with ultraviolet B. Nat Immunol 2007; 8:1095-1104.

MAST CELL APOPTOSIS AND SURVIVA V L

57

— † # @= †  \     ^      % # $  # N Engl J Med 2002; 346:1699-1705. 9. Bradding P, Walls AF, Holgate ST. The role of the mast cell in the pathophysiology of asthma. J Allergy Clin Immunol 2006; 117:1277-1284. 10. Theoharides TC, Conti P. Mast cells: the Jekyll and Hyde of tumor growth. Trends Immunol 2004; 25:235-241. 11. Soucek L, Lawlor ER, Soto D et al. Mast cells are required for angiogenesis and macroscopic expansion of Myc-induced pancreatic islet tumors. Nat Med 2007; 13:1211-1218. _ `$ † ƒ    #   @^       ! "  # {  $$   $  !  † _••—– __“€^_”€ 13. Kirshenbaum AS, Kessler SW, Goff JP et al. Demonstration of the origin of human mast cells from CD34

bone marrow progenitor cells. J Immunol 1991; 146:1410-1415. 14. Kitamura Y, Shimada M, Hatanaka K et al. Development of mast cells from grafted bone marrow cells in irradiated mice. Nature 1977; 268:442-443. 15. Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev 1997; 77:1033-1079. 16. Kitamura Y. Heterogeneity of mast cells and phenotypic change between subpopulations. Annu Rev Immunol 1989; 7:59-76. ” = < ƒ       $  =  "  \#    1966; 66:289-302. —    = < ƒ *#$   ! $    ‹$! {$   study of the growth of peritoneal mast cells in normal and athymic rats. Int Arch Allergy Appl Immunol 1985; 78:277-282. 19. Irani AA, Schechter NM, Craig SS et al. Two types of human mast cells that have distinct neutral protease compositions. Proc Natl Acad Sci USA 1986; 83:4464-4468. _• ~  @ >>      #   >   ‚  { Basic Principles and Clinical Correlates. 3rd ed.. Philadelphia: Lippincott Williams and Wilkins; 1999:97-117. _     @   †      #  ^! #^!   type in gastrointestinal mucosa of patients with defective T-lymphocyte function. J Immunol 1987; 138:4381-4386. 22. Friend DS, Ghildyal N, Austen KF et al. Mast cells that reside at different locations in the jejunum of mice infected with Trichinella spiralis exhibit sequential changes in their granule ultrastructure and chymase phenotype. J Cell Biol 1996; 135:279-290. 23. Lee YM, Jippo T, Kim DK K et al. Alteration of protease expression phenotype of mouse peritoneal mast cells by changing the microenvironment as demonstrated by in situ hybridization histochemistry. Am J Pathol 1998; 153:931-936. 24. Friend DS, Ghildyal N, Gurish MF et al. Reversible expression of tryptases and chymases in the jejunal mast cells of mice infected with Trichinella spiralis J Immunol 1998; 160:5537-5545. 25. Boyce JA. Mast cells: beyond IgE. J Allergy Clin Immunol 2003; 111:24-32; quiz 33. 26. Robinson DS, Hamid Q, Jacobson M et al. Evidence for Th2-type T helper cell control of allergic disease in vivo. Springer Semin Immunopathol 1993; 15:17-27. 27. Gibson PG, Allen CJ, Yang JP et al. Intraepithelial mast cells in allergic and nonallergic asthma. Assessment using bronchial brushings. Am Rev Respir Dis 1993; 148:80-86. _— \  †             $    @# €€Œ– •Œ€—€^€€“ 29. Garriga MM, Friedman MM, Metcalfe DD. A survey of the number and distribution of mast cells in the skin of patients with mast cell disorders. J Allergy Clin Immunol 1988; 82:425-432. 30. Malone DG, Wilder RL, Saavedra-Delgado AM et al. Mast cell numbers in rheumatoid synovial tissues. @  %# ‹$! $   #     $  {  therapy. Arthritis R Rheum 1987; 30:130-137. Œ   =   $           %#     `<¤ lymphoma. Br J Haematol. 2002; 119:122-124. 32. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267:1456-1462. 33. Vermeulen K, Van Bockstaele DR, Berneman ZN. Apoptosis: mechanisms and relevance in cancer. Ann Hematol 2005; 84:627-639. 34. Mahoney JA, Rosen A. Apoptosis and autoimmunity. Curr Opin Immunol 2005; 17:583-588. 35. Riedl SJ, Shi Y. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol 2004; 5:897-907. 36. Nagata S. Fas ligand-induced apoptosis. Annu Rev Genet 1999; 33:29-55. 37. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281:1309-1312. 38. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002; 2:647-656.

58

MAST CELL BIOLOGY

39. Adams JM, Cory S. Life-or-death decisions by the Bcl-2 protein family. Trends Biochem Sci 2001; 26:61-66. 40. Salvesen GS, Duckett CS. IAP proteins: blocking the road to death’s door. Nat Rev Mol Cell Biol 2002; 3:401-410. 41. Shiozaki EN, Shi Y. Caspases, IAPs and Smac/DIABLO: mechanisms from structural biology. Trends Biochem Sci 2004; 29:486-494. 42. Strasser A, Harris AW, Huang DC et al. Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J 1995; 14:6136-6147. 43. Li H, Zhu H, Xu CJ et al. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 1998; 94:491-501. 44. Huang DC, Strasser A. BH3-Only proteins-essential initiators of apoptotic cell death. Cell 2000; 103:839-842. 45. Chen L, Willis SN, Wei A et al. Differential Targeting of Prosurvival Bcl-2 Proteins by Their BH3-Only Ligands Allows Complementary Apoptotic Function. Mol Cell 2005; 17:393-403. 46. Kuwana K T, Bouchier-Hayes L, Chipuk JE et al. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell 2005; 17:525-535. 47. Cheng EH, Wei MC, Weiler S et al. BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 2001; 8:705-711. 48. Zong WX, Lindsten T, Ross AJ et al. BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak. Genes Dev 2001; 15:1481-1486. 49. Adams JM, Cory S. Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr Opin Immunol 2007; 19:488-496. 50. Baell JB, Huang DC. Prospects for targeting the Bcl-2 family of proteins to develop novel cytotoxic drugs. Biochem Pharmacol 2002; 64:851-863. 51. Kitamura Y, Go S. Decreased production of mast cells in S1/S1d anemic mice. Blood 1979; 53:492-497. 52. Kitamura Y, Go S, Hatanaka K. Decrease of mast cells in W/Wv mice and their increase by bone marrow transplantation. Blood 1978; 52:447-452. Œ   @# @@ \<     ^  &^# ^< $  &^#&^# mice as a model for investigating mast cell biology in vivo. Am J Pathol 2005; 167:835-848. 54. Finotto S, Mekori YA, Metcalfe DD. Glucocorticoids decrease tissue mast cell number by reducing the production of the c-kit ligand, stem cell factor, by resident cells: in vitro and in vivo evidence in murine systems. J Clin Invest 1997; 99:1721-1728. 55. Mekori YA, Oh CK, Metcalfe DD. IL-3-dependent murine mast cells undergo apoptosis on removal of IL-3. Prevention of apoptosis by c-kit ligand. J Immunol 1993; 151:3775-3784. 56. Iemura A, Tsai M, Ando A et al. The c-kit ligand, stem cell factor, promotes mast cell survival by suppressing apoptosis. Am J Pathol 1994; 144:321-328. 57. Juurikivi A, Sandler C, Lindstedt KA et al. Inhibition of c-kit tyrosine kinase by imatinib mesylate induces apoptosis in mast cells in rheumatoid synovia: a potential approach to the treatment of arthritis. Ann Rheum Dis 2005; 64:1126-1131. R 58. Möller C, Alfredsson J, Engström M et al. Stem cell factor promotes mast cell survival via inactivation of FOXO3a-mediated transcriptional induction and MEK-regulated phosphorylation of the proapoptotic protein Bim. Blood 2005; 106:1330-1336. 59. Biswas SC, Greene LA. Nerve growth factor (NGF) down-regulates the Bcl-2 homology 3 (BH3) domain-only protein Bim and suppresses its proapoptotic activity by phosphorylation. J Biol Chem 2002; 277:49511-49516. “• ƒ ‚ †  `     !  # =‚_  \#% \  Phosphorylation and Proteasome-dependent Degradation of the BH3-only Protein, Bim. J Biol Chem 2003; 278:18811-11816. 61. Luciano F, Jacquel A, Colosetti P et al. Phosphorylation of Bim-EL by Erk1/2 on serine 69 promotes its degradation via the proteasome pathway and regulates its proapoptotic function. Oncogene 2003; 22:6785-6793. 62. Alfredsson J, Puthalakath H, Martin H et al. Proapoptotic Bcl-2 family member Bim is involved in the control of mast cell survival and is induced together with Bcl-X(L) upon IgE-receptor activation. Cell Death Differ 2005; 12:136-144. 63. Ekoff M, Kaufmann T, Engstrom M et al. The BH3-only protein Puma plays an essential role in cytokine deprivation induced apoptosis of mast cells. Blood 2007; 110:3209-3217. 64. Lindsten T, Ross AJ, King A et al. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol Cell 2000; 6:1389-1399. “ $ @  *$  *$  &   †"^   %# # #    germ cell death. Science 1995; 270:96-99.

MAST CELL APOPTOSIS AND SURVIVA V L

59

““ ‚# >@ ƒ *   &      †<  †"  $  #   # homeostasis. Nat Immunol 2002; 3:932-939. 67. Maurer M, Tsai M, Metz M et al. A role for Bax in the regulation of apoptosis in mouse mast cells. J Invest Dermatol 2000; 114:1205-1206. 68. Karlberg M, Ekoff M, Labi V et al. Pro-apoptotic Bax is the major and Bak an auxiliary effector in cytokine deprivation-induced mast cell apoptosis. Cell Death Dis 2010;e43. “€   ž ˆ¤@ ƒ ˆ¤‚ ƒ      †^_^ $  ! independently of the cytochrome c/Apaff 1/caspase-9 apoptosome. Nature 2002; 419:634-637. ”•   ž $ * ˆ¤‚ ƒ   ^    ^€   ‹$   < withdrawal-induced apoptosis of mast cells but dispensable for their functional and clonogenic death. Blood 2006; 107:1872-1877. 71. Bouillet P, Metcalf D, Huang DC et al. Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis and to preclude autoimmunity. Science 1999; 286:1735-1738. 72. Coultas L, Strasser A. The role of the Bcl-2 protein family in cancer. Semin Cancer Biol 2003; 13:115-123. 73. Hamasaki A, Sendo F, Nakayama K et al. Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene. J Exp Med 1998; 188:1985-1992. 74. Xiang Z, Ahmed AA, Moller C et al. Essential role of the prosurvival bcl-2 homologue A1 in mast cell survival after allergic activation. J Exp Med 2001; 194:1561-1569. ” ‚<  >ƒ `   < †   ^   $   ^   lethality. Genes Dev 2000; 14:23-27. 76. Motoyama N, Wang F, Roth KA et al. Massive cell death of immature hematopoietic cells and neurons in †^"^    €€– _“”•“^• 77. Nakayama K, Nakayama K, Negishi I et al. Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease and lymphocytopenia. Proc Natl Acad Sci USA 1994; 91:3700-3704. 78. Moller C, Karlberg M, Abrink M et al. Bcl-2 and Bcl-XL are indispensable for the late phase of mast cell development from mouse embryonic stem cells. Exp Hematol 2007; 35:385-393. 79. Cohen-Saidon C, Nechushtan H, Kahlon S et al. A novel strategy using single-chain antibody to show the importance of Bcl-2 in mast cell survival. Blood 2003; 102:2506-2512. 80. Hartmann K, Wagelie-Steffen AL, von Stebut E et al. Fas (CD95, APO-1) antigen expression and function in murine mast cells. J Immunol 1997; 159:4006-4014. 81. Berent-Maoz B, Piliponsky AM, Daigle I et al. Human mast cells undergo TRAIL-induced apoptosis. J Immunol 2006; 176:2272-2278. 82. Kobayasi T, Asboe-Hansen G. Degranulation and regranulation of human mast cells. An electron microscopic study of the whealing reaction in urticaria pigmentosa. Acta Derm Venereol 1969; 49:369-381. 83. Dvorak AM, Schleimer RP, Lichtenstein LM. Morphologic mast cell cycles. Cell Immunol 1987; 105:199-204. 84. Kawakami T, Galli SJ. Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2002; 2:773-786. 85. Kitaura J, Xiao W, Maeda-Yamamoto M et al. Early divergence of Fc epsilon receptor I signals for receptor up-regulation and internalization from degranulation, cytokine production and survival. J Immunol 2004; 173:4317-4323. 86. Yoshikawa H, Nakajima Y, Tasaka K. Glucocorticoid suppresses autocrine survival of mast cells by inhibiting IL-4 production and ICAM-1 expression. J Immunol 1999; 162:6162-6170. 87. Kalesnikoff J, Huber M, Lam V et al. Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity. 2001; 14:801-811. —— † ^ } †   <$    *‚ƒ    #$   # {$ of IgE-dependent activation. Allergy 2008; 63:333-340. 89. Xiao W, Nishimoto H, Hong H et al. Positive and negative regulation of mast cell activation by Lyn via the FcepsilonRI. J Immunol 2005; 175:6885-6892. 90. Yamasaki S, Ishikawa E, Kohno M et al. The quantity and duration of FcRgamma signals determine mast cell degranulation and survival. Blood 2004; 103:3093-3101. 91. Ekoff M, Strasser A, Nilsson G. FcepsilonRI aggregation promotes survival of connective tissue-like mast cells but not mucosal-like mast cells. J Immunol 2007; 178:4177-4183. €_  # š  #      Ÿ$  {    #  $  patients with allergic rhinitis and normal subjects. J Allergy Clin Immunol 1995; 95:716-725. 93. Möller C, Xiang Z, Nilsson G. Activation of mast cells by immunoglobulin E-receptor cross-linkage, but not through adenosine receptors, induces A1 expression and promotes survival. Clin Exp Allergy 2003; 33:1135-1140.

60

MAST CELL BIOLOGY

€’     @ ~  =^  ! $ $ !!  †{^ "   #$ mast cells but not basophils. Allergy 2006; 61:1040-1046. 95. Alfredsson J, Moller C, Nilsson G. IgE-receptor activation of mast cells regulates phosphorylation and expression of forkhead and Bcl-2 family members. Scand J Immunol 2006; 63:1-6. 96. Herold MJ, Zeitz J, Pelzer C et al. The stability and anti-apoptotic function of A1 are controlled by its C terminus. J Biol Chem 2006; 281:13663-13671. 97. Valent P, Akin C, Sperr WR R et al. Mastocytosis: pathology, genetics and current options for therapy. Leuk Lymphoma 2005; 46:35-48. 98. Longley BJ, Tyrrell L, Lu SZ et al. Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm. Nat Genet 1996; 12:312-314. 99. Longley BJ, Jr., Metcalfe DD, Tharp M et al. Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis. Proc Natl Acad Sci USA 1999; 96:1609-1614. •• ~ ` &   ˆ# @        $  #    # protooncogene c-kit in peripheral blood mononuclear cells of patients who have mastocytosis with an associated hematologic disorder. Proc Natl Acad Sci USA 1995; 92:10560-10564. • #  > " ž  <         †   $ $  in neoplastic mast cells: role of KIT D816V and effects of various targeted drugs. Blood 2009; 114:5342-5351. •_ #  >  #  " ž      @ƒ   !     mast cells in systemic mastocytosis: inhibition of mast cell survival by MCL1 antisense oligonucleotides and synergism with PKC412. Blood 2007; 109:3031-3041. 103. Cervero C, Escribano L, San Miguel JF et al. Expression of Bcl-2 by human bone marrow mast cells and its overexpression in mast cell leukemia. Am J Hematol 1999; 60:191-195. 104. Baghestanian M, Jordan JH, Kiener HP et al. Activation of human mast cells through stem cell factor receptor (KIT) is associated with expression of bcl-2. Int Arch Allergy Immunol 2002; 129:228-236. 105. Hartmann K, Artuc M, Baldus SE et al. Expression of Bcl-2 and Bcl-xL in cutaneous and bone marrow lesions of mastocytosis. Am J Pathol 2003; 163:819-826. 106. Oltersdorf T, Elmore SW, Shoemaker AR R et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 2005; 435:677-681. 107. van Delft MF, Wei AH, Mason KD et al. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and   $  ! †<†"  ^  $ } @ @ _••“– •Œ—€^Œ€€ 108. Konopleva M, Contractor R, Tsao T et al. Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia. Cancer Cell 2006; 10:375-388. •€ *#  š      {$  †^_     # $   small-cell lung cancer cell lines to ABT-737. Cancer Res 2007; 67:1176-1183. 110. Chen S, Dai Y, Harada H et al. Mcl-1 down-regulation potentiates ABT-737 lethality by cooperatively inducing Bak activation and Bax translocation. Cancer Res 2007; 67:782-791. 111. Lin X, Morgan-Lappe S, Huang X et al. ‘Seed’ analysis of offf target siRN R As reveals an essential role of Mcl-1 in resistance to the small-molecule Bcl-2/Bcl-XL inhibitor ABT-737. Oncogene 2007; 26:3972-3979. 112. Smits C, Czabotar PE, Hinds MG et al. Structural plasticity underpins promiscuous binding of the prosurvival protein A1. Structure 2008; 16:818-829. 113. Herman MD, Nyman T, Welin M et al. Completing the family portrait of the anti-apoptotic Bcl-2 proteins:    $$   #$ †{^  " %# † =† ƒ _••—– —_Œ€•^Œ€’

SECTION III

MAST CELL ACTIVATION

CHAPTER R5 REGULATORS OF Ca2 SIGNALING IN MAST CELLS: Potential Targets for Treatment of Mast Cell-Related Diseases? `^*   #  †!| Laboratory of Molecular Immunology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA      0  &  &  

Abstract:

A calcium signal is essential for degranulation, generation of eicosanoids and optimal production of cytokines in mast cells in response to antigen and other stimulants. The signal is initiated by phospholipaseC-mediated production of inositol 1,4,5-trisphosphate resulting in release of stored Ca2 from the endoplasmic reticulum (ER) and Golgi.   #   ! {$"  " $ @2 , usually referred to as store-operated calcium entry (SOCE), through the interaction of the Caa2 -sensor, stromal interacting molecule-1 (STIM1), in ER R with Orai1(CRACM1) and transient receptor potential canonical (TRP R C) channel proteins in the plasma membrane (PM). This interaction is enabled by microtubular-directed reorganization of ER R to form ER/ R PM contact points or “punctae” in which STIM1 and channel proteins colocalize. The $ {$"  @2 replenishes Ca2 stores and sustains elevated levels of cytosolic Ca2 ions-the obligatory signal for mast-cell activation. In addition, the signal can acquire spatial and dynamic characteristics (e.g., calcium puffs, waves, oscillations) #      $ $$ *#  #!    regulation of Ca2 {$" # $# *\^ @2 -pumps and ion exchangers in mitochondria, ER R and PM. As discussed in this chapter, studies in mast cells revealed much about the mechanisms described above but little about allergic and autoimmune diseases although studies in other types of cells have exposed genetic defects that lead to aberrant calcium signaling in immune diseases. Pharmacologic agents that inhibit or activate the regulatory components of calcium signaling in mast cells are also discussed along with the prospects for development of novel SOCE inhibitors #   !    #   {  ^  

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media.

62

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

63

INTRODUCTION The generation of a calcium signal is critical for activation of mast cells and blood basophils. This signal results from receptor-mediated activation of phospholipase (PL) C and the associated production of inositol 1,4,5-trisphosphate (IP3) which induces release of Ca2 from stores in the endoplasmic reticulum (ER) and Golgi through Ca2 -conducting IP3-receptors (IP3R). Depletion of the Ca2 ^  ! {$"  external Ca2 , a process commonly referred to as store-operated calcium entry or SOCE (reviewed in reference 1). One well characterized mechanism of Ca2 {$" ! ˆ@= is the Ca2 -selective calcium-release activated calcium current (IICRAC™     RBL mast cells by Hoth and Penner.2,3 *# {$"  @2 allows reuptake of Ca2 into ER through a sarco/endoplasmic Ca2 -ATPase (SERCA) pump and thereby replenishes ER stores and sustains increases in concentration of cytosolic Ca2 ([Ca2 ]cyt), the essential signal for mast cell activation. [Ca2 ]cyt is also dynamically regulated by extrusion of Ca2 from the cell by a Ca2 -ATPase (PMCA) pump and ion-exchange transporters in #    *#     $   # $  within the cell is further shaped by mitochondria which possess specialized mechanisms for Ca2 ^ $<  {$"   ##   $^$  ˜ ™ *# spatial and temporal characteristics of the calcium signal, which may include calcium “puffs”, “waves” and “oscillations”, are thought to encode sub-signals for different cellular functions.    =^   #   $  $ PLC-mediated calcium signals. These stimulants include the growth factor KIT ligand, also referred to as stem cell factor (SCF),4 as well as agonists of G protein-coupled receptors such as adenosine,5-7 PGE2,8,9 the formyl peptide fMetLeuPhe,10 platelet activating factor,10 and C3a/C5a.11 Some mast cell activating ligands such as microbial Toll-like receptor (TLR) ligands and IL-33 can elicit modest production of cytokines without mobilizing calcium or causing degranulation but this production of cytokines is markedly enhanced by costimulation of cells with antigen which elicits synergistic costimulatory signals though calcium.12 Calcium signals thus enable degranulation,13,14 activation of PLA2 for the production of the eicosanoids,15 activation of the Ca2 /calcineurin/nuclear factor of activation of T cells (NFAT) pathway for transcription of cytokine genes,12 or Ca2 -dependent cytoskeletal remodeling for chemotaxis (Fig. 1).11 The description of ICRAC prompted a prolonged search for an elusive ICRAC channel   ˜@‚@™ *#    @‚@ %       # Ca2 -channel protein Orai1 (also known as CRACM1) and its activating ER R calcium-sensor, stromal interaction molecule-1 (STIM1). Before then, the nonselective Ca2 -conducting transient receptor potential canonical (TRPC) proteins were initially considered as candidates for CRAC but none of the TRPCs exhibited the exact characteristics or Ca2 -selectivity off ICRAC. Because expression of both STIM116,17 and Orai118-20 reconstituted ICRAC   21-23 in various types of cells including mast cells, the pendulum of thinking has swung towards the notion that the activating interaction of STIM1 with Orai1 is the underlying mechanism for SOCE. There remains, nevertheless, an accumulating body of evidence that TRPCs participate in SOCE, possibly cooperatively with STIM1 and Orai1, in mast cells and other types of cells.1 As discussed in the next section, the participation of TRPCs would be consistent with observations several decades ago that other divalent metal ions are taken up by and can substitute for Ca2 in the activation of mast cells.

64

MAST CELL BIOLOGY

Figure 1. @$ {$"  $     !  #   @2 through IP3 receptors (IP3R) from Ca2 stores in ER (as depicted), Golgi and nuclear membrane (not depicted) into the cytosol following Fc¡RI (R) aggregation by antigen (Ag) and the associated production of IP 3 by PLCa. Ca2 released in close proximity to mitochondria is rapidly taken up through the mitochondrial uniporter (U) which operates only with high [Ca2 ]cyt (1–10 +M) in restricted spaces (as shown) or during large oscillations in [Ca2 ]cyt. In this manner, mitochondria can accelerate depletion of Ca2+     #     # $  =%#  %  @2 diffuses through the cytosol to elevate [Ca2 ]cyt.. Letm1 Ca2 /H antiporter (L) allows slow entry of Ca2 into mitochondria at relatively low [Ca2 ]cyt (100 nM) and thus enables mitochondria to sense small increases in [Ca2 ] 2

along cyt as a signal for increased mitochondrial activity. Letm1 is bidirectional and can extrude Ca with a Na /Ca2 exchanger NCX (N) when the mitochondrial Ca2 -load is high. Depletion of the Ca2

stores causes formation of contact points (punctae) that bring the ER Ca2 -sensor STIM1 (not shown but see Fig. 2) and channel proteins (Orai and TRPC) into close proximity to activate Ca2 {$" *# {$" # =‚ @2 stores via the ATP-dependent SERCA pumps (SERCA 2b or SERCA3), sustains elevation of [Ca2 ]cyt and continuous recycling of Ca2 as long as IP3   $ {$"  Ca2 ! ˆ   *‚\@  ! $  {$"  ~ via TRPM4 and positively regulated by {$"   via IKCa3.1 which, respectively, depolarize or repolarize the membrane potential. [Ca2 ]cyt is held in dynamic equilibrium by extrusion of Ca2 through the Na /Ca2 exchangers (N) as well as the ATP-dependent PMCA pump. Candidates for a putative “leak” channel (?) in ER are discussed in the text. Also shown are the responses evoked by calcium signaling in mast cells.

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

65

 # #  %    #   $  $      just noted. Subsequent sections provide descriptions of various organelles that regulate calcium signaling in mast cells. Although the contributions of ER, mitochondria and the plasma membrane are described separately for convenience of narrative, it should  #} # #  $   $      #     # #    ˜ ™ *#     $!  describe the functional outputs encoded by the calcium signal, the possible relevance of genetically-related defects in calcium signaling to mast cell related diseases and novel SOCE inhibitors developed for the purpose of treating allergic and autoimmune diseases.

EARLY AND RECENT OBSERVATIONS ON CALCIUM HOMEOSTASIS IN MAST CELLS: THE UNRESOLVED ISSUES

   # %   #  "     $  the study of Ca2 -dependent secretion when it was discovered that extracellular Ca2 was required for anaphylactic release of histamine from human leukocytes,24 rabbit basophils,25 and rat peritoneal mast cells.26 By then, histamine was shown to be a constituent of granules secreted from mast cells.27 Subsequent studies revealed that Sr2 or Ba2 could substitute for Ca2 in supporting histamine release from rat peritoneal mast cells26,28 and that uptake of 45Ca2 and 89Sr2 correlated with the extent of this release.28,29 Such release, whether supported by Ca2 or Sr2 , was effectively blocked by low concentrations of La3 .30,31 The conclusions drawn from these studies was that mast cell degranulation was   $ {$"  @2 through “Ca2 channels” which can convey Sr2 .28 Later studies with Ca2 ^! {$     45Ca2 in cultured RBL-2H3 cells indicated that degranulation was absolutely dependent on an increase in [Ca2 ]cyt from ¾0.1 to ¾1 +M13 through release of Ca2 from intracellular stores32-35  {$" of extracellular Ca2 .13   #   $ # {$"       2 and other divalent metal ions.36 {$" %   $ $<  @2 into IP3-sensitive and mitochondrial pools37,38 wherein intracellular Ca2 increased from approximately 0.5 mM to about 2.5 mM which then decreased as [Ca2 ]cyt subsided.32 {$" %     #    #     $ # $# {$"   ,32,39 and was counterbalanced by extrusion of Ca2 from the cell by an ATP-dependent mechanism.32,40 $  #      =‚  # $   \3-releasable Ca2

and IP3R41 as the channel for Ca2 -release.42 Reuptake of Ca2 into ER R was blocked by thapsigargin which was found to be selective inhibitor of SERCA.43,44 This action of thapsigargin results in spontaneous loss or “leakage” of Ca2 from the ER R store45,46 by   $  #   # =‚  %##  #   \3, invariably leads to entry of Ca2 into the cell. This feature led to the proposal by Putney in 1986, of “capacitative calcium entry” now generally referred to as SOCE,47 a process that is relevant to mast cells.38,48 As noted in the previous section, the characterization of ICRAC in mast cells by Hoth and Penner2,3 $ $  #    # STIM1/Orai1 as the operational components of ICRAC. ~! # # ##   {$" #  @2 and other divalent metal ions in mast cells are contrary to the known features of ICRAC or Orai proteins. We have proposed on the basis of knockdown and overexpression studies in RBL-2H3 cells that TRPC5, which can convey Sr2    %# *   ˆ   # {$"

66

MAST CELL BIOLOGY

of Ca2 and, as a consequence, allow entry of Sr2 .49 An alternative proposal based on studies in RBL-2H3 cells and bone marrow-derived mast cells (BMMC) is that TRPCs, particularly TRPC1, initiate localized Ca2 puffs that potentiate IP3R-mediated Ca2 -release from nearby ER R stores.50 This potentiation leads to the generation of a calcium wave and ultimately oscillations, phenomena commonly associated with SOCE. As discussed in detail later, studies in other types of cells suggest that some TRPCs, including TRPC1 and TRPC5, form complexes with Orai proteins and STIM1to create SOCE channels whose conductances ((IISOC) differ from those of ICRAC. The role of TRPCs in calcium signaling is controversial. One view is that CRAC channels derived from STIM1 and Orai1 are the exclusive SOCE channel. The other is that STIM1, TRPCs and Orai proteins also form SOCE channels with varying degrees of selectivity (e.g., refs. 51,52). This chapter { $  # #  #     

+%+&+&+(% (^ &=$ +) + % '+  +3R AND SPHINGOSINE KINASES (SKs) Receptor-Mediated Activation of PLCa or PLC` Results in SOCE A role for PLC was evident from early studies with RBL-2H3 cells in which correlations were noted in the production of inositol phosphates, increase in [Ca2 ]cytt and degranulation.53 The PLC isoforms involved differed according to the type of stimulant; PLCa with antigen54,55 and PLC` with ligands to G protein-coupled receptors.10,56 Stimulation through either Fc¡RI or G protein-coupled receptors resulted in release of Ca2 from a common IP3/thapsigargin-sensitive pool which was assumed to be in ER.5,38,57 Recently, stochastic modeling using three-dimensional constructs of electron-microscopic images  ‚†ƒ^_`Œ   %# $    % %# #     Ca2 from ER R through activation of IP3Rs by IP3.58 RBL-2H3 cells, BMMC and human peripheral blood-derived mast cells express both PLCa1 and PLCa2.59-61 PLCa1 resides primarily in cytosol of RBL-2H3 cells but then migrates  #    %#   }    ^ #   ${  antigen stimulation.59 PLCa2, in contrast, is associated with Golgi and plasma membranes and remains so after stimulation. Both isoforms are tyrosine phosphorylated and thus activated, following antigen stimulation. The pathways leading to PLCa phosphorylation are complex and are not entirely clear. Induction of Fc¡RI aggregation by antigen initiates phosphorylation cascades that involve the Src kinases Lyn and Fyn as well as Syk tyrosine kinase, phosphorylation of adaptor proteins, recruitment of additional signaling molecules by these adaptors and ultimately production of IP3 by PLCa. The adaptor proteins, linker  !  *  ˜ƒ*™  `_^ $<^     ”“ KDa (SLP-76) are critical for recruitment of PLCa1 and calcium signaling (for a more detailed account see reference 62). Another essential component of mast cell activation is phosphoinositide 3-kinase (PI3K) whose activity is regulated largely by Fyn through the adaptor protein, Grb2-associated binder-like protein 2 (Gab2).63 It is unclear, however, whether the PLCs are activated separately through different pathways. Differences have been noted in RBL-2H3 cells where phosphorylation of PLCa1, but not PLCa2, is suppressed by the PI3K K inhibitor wortmannin.59 In these cells, PLCa1 was assumed to play the predominant role as both antigen-induced IP3 production and degranulation are also inhibited by wortmannin. In BMMC and human peripheral blood-derived mast cells, the

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

67

initial activation of PLCa1 occurs independently of PI3K.61 Indeed the phosphorylation and activation of both PLCa1 and PLCa2 are resistant to wortmannin, are still apparent in BMMC that lack p85 PI3K K subunits and appear to precede the activation of PI3K. Wortmannin partially inhibits degranulation leaving the possibility that PI3K K mediates later signals for degranulation.61 The differential distribution of the two isoforms of PLCa in RBL-2H3 cells could result theoretically in calcium signals with different signatures. Whether the PLC isoforms are regulated differently and play complimentary roles in calcium signaling in mast cells are issues that require further investigation. The pattern of expression of PLC` isoforms in mast cells varies. Of the three known PLC` isoforms, only PLC`3 is expressed in RBL-2H3 cells56,64 whereas PLC`2 and PLC`3 are expressed in BMMC.9 PLC` is activated through receptors that engage either the pertussis-sensitive G protein, Gi,9 or the pertussis- and cholera- toxin insensitive G protein, Gq.6 Receptors that operate through Gs and adenylate cyclase are generally inhibitory. Some of the Gi/q-linked receptors undergo rapid desensitization such that IP3 production  $       $     $ 8,65 However, ligands to such receptors can markedly potentiate the functional responses to antigen because of synergistic signaling interactions provoked by the sustained calcium signal induced by antigen.65 *    \ <\? +# \q  \| # %        *#$#$  ——’   # for the mythological riddle of the Sphinx.66,67 In some respects this is still true for calcium signaling.1,68,69 In the modern era, the SK K product, sphingosine 1-phosphate (S1P), was proposed to complement the actions of IP3 in promoting SOCE, degranulation,70,71 and the production of eicosanoids and cytokines72 in mast cells. Both IP3 and S1P were considered essential for release of Ca2 from intracellular stores70 with S1P promoting transient release and IP3 a more sustained release coupled to Ca2 -entry.71 However, a subsequent report suggests that S1P regulates primarily Ca2 -entry rather than release.72 Another ambiguity is that SK1 was claimed to be the predominant regulator of calcium signaling in one study71 and SK2 in another.72 An uncertainty is that S1P is released from stimulated mast cells73 and could act in an autocrine manner74 to increase [Ca2 ]cytt via PLC`-coupled S1P receptors.75 Potential upstream regulators of the SK/ K Ca2 -pathway 76 include the Src kinases Lyn and Fyn, PLD1 and clathrin which is thought to facilitate transfer of the lipophilic S1P from the plasma membrane to ER.77 Unlike IP3, S1P is not freely diffusible in the cytosol68 and the Ca2 -channel(s) targeted by S1P have yet to be  69 Regardless of these unresolved issues, there is corroborative and extensive evidence that S1P and related sphingolipids are critical for mast-cell driven allergic reactions74,78-80 as discussed in detail in the Chapter 8 in this book. Function and Regulation of IP3R R in ER IP3Rs are of three subtypes (designated 1, 2 and 3) and are expressed predominantly in the ER R and to a lesser extent in nuclear reticulum (NR) and Golgi.81-85 The IP3Rs are usually restricted to particular subcellular locations. In RBL-2H3 cells, IP3R1 and IP3R2 are located in ER R and NR whereas IP3R3 is located elsewhere.86,87 RBL-2H3 cells express ¾14,000 tetrameric IP3Rs with IP3R2 being the predominant form (70% of total) and IPR1 and IPR3 constituting 10% and 20% of total IP3Rs.87 As in other types

68

MAST CELL BIOLOGY

of cells,81 stimulation of RBL-2H3 cells results in redistribution of IP3R from a diffuse to clustered pattern in ER R and NR.87 The relevance of clustering is unclear but it could signify association with sites of IP3 production58 or formation of signaling complexes, sometimes referred to as signalosomes, which may contain IP3R, TRPCs, receptors for activated C-kinase-1 (RACK1), STIM1 and Orai1 (see ref. 88 and citations therein). IP3Rs form functional Ca2 channels only after oligomerization to form homo- or hetero-tetramers.85,89 Interestingly, the same is true for Orai and TRPC proteins which also form functional channels only after tetramerization. Each IP3R subunit consists of a cytosolic N-terminal IP3-binding domain, a large intermediate modulatory domain and a C-terminal helical transmembrane-spanning domain which, in the tetrameric state, form part of the Ca2 channel. The IP3-binding domain lies in close proximity to the #       # #   $   # 85,89 The intermediate domain contains binding sites for Ca2 , calmodulin and other factors that regulate IP3R function.81,83,85 All three IP3Rs exhibit biphasic responses to Ca2 in which IP3R activity is enhanced or diminished at low or high [Ca2 ]cytt respectively90 by mechanisms that are still conjectural.1 Other regulatory factors include RACK1 and scaffolding proteins that may allow targeting of IP3R to strategic sites and formation of signaling complexes.83,85 IP3Rs are phosphorylated by multiple serine/threonine protein kinases creating potential positive feed-back loops to enhance Ca2 -release.85,91 The Src tyrosine kinases, Fyn in T-cells92 and Lyn in B cells,93 also phosphorylate and positively regulate IP3R but it is not known if this occurs upon activation of these same kinases in mast cells. Many aspects of the regulation of IP3R activity remain unclear as described in several recent reviews.83,84,89,91 IP3-SENSITIVE INTRAC R ELLULAR Ca2 -STORES AND COUPLING TO SOCE CHANNELS IP3-Releasable Ca2 Stores In addition to ER, IP3-releasable Ca2 stores are present in NR94 and Golgi.95 The ER R and NR form a contiguous Ca2 -pool of ¾0.5 mM but Golgi forms a separate pool of ¾0.3 mM Ca2 . Initial increases in [Ca2 ]cytt are primarily due to release of Ca2 from =‚   %#     ~‚       # $ 94 All three structures contain the thapsigargin-sensitive SERCA pump that ensures recapture of cytosolic Ca2 against a high concentration gradient (also see following section). The Golgi membranes contain, in addition, a thapsigargin-insensitive Ca2 -ATPase (SPCA) pump. The individual contributions of Golgi and ER R to the calcium signal in mast cells are unknown but several IP3-releasable pools are discernable in RBL-2H3 cells.96 Two of these are thapsigargin-sensitive of which only one is directly linked to ICRAC. The other is a thapsigargin-insensitive store which is presumed to be dependent on a calcium *\ # # =‚@     # @2 store. Compartmentalization of Ca2

stores in ER/Golgi R is also suggested from the differential effects of IP3, thapsigargin and ionomycin on ICRAC dynamics in the RBL1 mast cell line.97 The nature of these pools or compartments is unclear but there is evidence that, in some types of cells, Ca2 can diffuse throughout the lumen of ER R and depletion of the entire ER store is required for SOCE whereas in other types, such as polarized liver cells, diffusion of Ca2 is restricted        =‚  $   ! ˆ@=98 It is likely that

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

69

SOCE is activated in RBL-2H3 cells by depletion of ER R stores near the cell periphery as SOCE can be activated by just 1 nM thapsigargin,38 a concentration that is known to localize and act only in regions of ER R in close proximity to the plasma membrane.99 This concentration of thapsigargin does not induce degranulation whereas more conventional concentrations of thapsigargin (e.g., 1 +M) produce a global depletion of ER R stores and degranulation.38,99 Actin and microtubular interactions direct ER R motility which is essential for regulation of calcium signaling and other cellular functions.100 This motility, as we shall discuss later, permits functional contacts of ER R with Ca2 channels in the plasma membrane. Depolymerization of microtubules or inhibition of the microtubular motors, kinesin or dynein, reduces SOCE.101,102 Unintended effects of pharmacologic agents on ER R motility and calcium signaling should be considered in studies of mast cell activation and function. Ca2 )>  }>~  $* Uptake and “leakage” of Ca2 across the ER R membrane appear to be dynamically balanced in resting RBL-2H3 cells.103 The “Ca2 leak” from ER R is apparent by a relatively slow increase in [Ca2 ]cytt in the absence of external Ca2 following blockade of SERCA by thapsigargin. Subsequent provision of external Ca2 results in a rapid and substantial increase in [Ca2 ]cytt which is the classic hallmark of SOCE. The number of SERCA channels, as determined by thapsigargin binding, has been estimated as 1.6 million/ RBL-2H3 cell.103 Of the three known SERCA proteins and their spliced variants, only SERCA2b and SERCA3 are found in mast cells104 where they codistribute with IP3R1, IP3R2 and calreticulin on density gradients.86 IP3R3 is the anomaly in that it does not appear to be associated with ER R or the SERCAs.86 The SERCA proteins transport two 2

Ca   # *\ $ $ %##     $    Ca2   #    $     =‚ %#   $   [Ca2 ]cytt when ER is in close proximity to SOCE channels.105 SERCA activity also enables Ca2 oscillations in cells.106 The SERCAs are regulated directly by free Ca2 within ER.107 The Ca2 sensing mechanism, however, is uncertain but suggested mechanisms include interactions with calreticulin and calnexin,108 STIM1,109,110 and presenilins.111 Calreticulin and calnexin reside in the ER R lumen and ER membrane respectively and are interacting proteins that are best recognized for their role in protein folding. Both proteins as well as the phosphorylation of calnexin are critical for maintenance of SERCA-dependent Ca2 oscillations in Xenopus oocytes.108,112 It is unknown whether similar mechanisms exist in mast cells but it is notable that calnexin is prominently phosphorylated in antigen-stimulated RBL-2H3 cells.113 The nature of the ER R “Ca2 -leak” protein is still a matter of debate (hence the question mark in Fig. 1). Proposed candidates include the presenilins,114,115 the translocon complex,116-118 and pannexin-1.119 The presenilins were reported to form Ca2 -permeable channels in bilayer vesicles whereas mutant presenilins that are thought to be responsible for enhanced calcium signaling in Alzheimer’s disease lacked such activity.114,115 However, others report no abnormality in ER Ca2 “leakage” in cells from such patients but have noted enhanced IP3-mediated release.120 The aberrant calcium signaling in Alzheimer’s disease has been attributed to interactions of constitutively active mutant presenilins with IP3Rs.121 The evidence in total does suggest a role for the presenilins in regulating Ca2 homeostasis in part because of the links to Alzheimer’s disease and the presumed interactions of presenilins with SERCA111 and IP3R.122 Although presenilins

70

MAST CELL BIOLOGY

are expressed and possibly contribute to calcium signaling in mast cells, their role has yet to be determined.1 Translocon, a protein-conducting channel in ER, can serve as a “leak pathway” for Ca2 117,123 and can activate SOCE116,124 when not engaged in protein synthesis.125 However, contrary data has led to the conclusion that it is normally closed and is irrelevant to the physiological calcium leak mechanisms.118 The pannexins, a recently described family of ubiquitous molecules analogous to gap junction connexins, form not only intercellular gap junction channels but also Ca2 -permeable channels in ER. Overexpression and knockdown studies implicate pannexin1 as a mediator of “Ca2 -leak” and an activator of SOCE “in addition to any other potential leak mechanism.”119 Whether one or more mechanisms actually exist, leakage has the intrinsic properties of an ion #  "# " {$"  @2 from ER R which shuts off once calcium stores have reached about 7% of their normal level.126 Leakage is also in dynamic balance with ER R uptake.58 & $* * 2 @ &+ q  &+ | The absolute requirement for STIM1 and Orai1 for activating ICRAC, calcium signaling, degranulation and cytokine production in mast cells has been demonstrated in BMMC  !   *   ˆ    127,128 These mice also have attenuated IgE-dependent allergic responses. Both isoforms of STIM are known regulators of SOCE in a variety of cell types.16,17,20 Both isoforms are highly homologous and contain an N-terminal EF hand along with a sterile _-motif (SAM) located in the ER R lumen, a single transmembrane domain and protein-interacting domains in their lumenal and cytoplasmic portions.16,17,129 *#    # =^   @2 is appropriately set to sense the concentration of free Ca2 in ER R ([Ca2 ]ER) and state of depletion of ER 2

130,131 Ca -stores. When ER Ca2 stores are full, STIM1 is diffusely distributed throughout the ER microtubular network101 but as these stores are depleted, STIM1 oligomerizes and migrates to peripheral ER R “punctae” in close proximity to the plasma membrane where it can activate ICRAC.16,22,132 Mutating the STIM1 EF-hand results in constitutive localization of STIM1 in “punctae” and activation of ICRAC independently of store-depletion.132,133 Collectively these studies imply that on store depletion dissociation of Ca2 from the EF-hand, which is mimicked by mutation of the EF-hand, results in oligomerization and relocation of STIM1. Rearrangement of ER R with formation of “punctae” containing colocalized STIM1 and Orai1 is also evident in antigen-stimulated RBL-2H3 cells.134 Maximal colocalization coincides with the initial spike in [Ca2 ]cytt with rapid reversal upon decay in [Ca2 ]cyt     =‚   The generally accepted scenario from studies in RBL-2H3134-136 and other types of cells is that coiled-coil domains in STIM1 and Orai1 and the electrostatic interactions between these two molecules are critical for the association of these two molecules and the activation of ICRAC.1 The oligomerization and migration of STIM1 to the cell periphery is dependent on a coiled-coil and another domain (aa 233-450, human numbering) in the cytoplasmic C-terminal region of STIM1.135,137 Functional interaction with Orai1 is dependent on a minimal domain (aa 342-448) in STIM1 called the CRACactivating domain (CAD)138 which   #   !  ‹$ ˜ Œ—_^Œ—”™ #  $   ! ˆ@= in RBL-2H3 cells.136 It is postulated that neutralization of a C-terminal acidic coiled-coil domain of Orai1134,139 by CAD transmits a gating signal for SOCE. In support of this idea, amphiphilic molecules such as D-sphingosine and N,NN dimethylsphingosine inhibit not

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

71

only IP3-mediated ICRAC140 but also the FRET-monitored interaction of STIM1 with Orai1 and Ca2 ^{$"134 The proposed model was that the positively charged sphingosines, %## {  #  $   #    $ } # ˆ   residues resulting in Orai1 homo-oligomerization and preclusion of STIM1. Although #     $  ˆ@=  # # "$    role for SKs because D-sphingosine is a substrate and N,NN dimethylsphingosine is an inhibitor of SK, yet both have the same effect. STIM1 also interacts with various TRPC channels, either individually or as heteromeric combinations with Orai,52,141,142 and may do so though intramolecular electrostatic interactions between complementary positively-charged residues in STIM1 and negatively-charged residues in TRPC1 or TRPC3. However, it is claimed that the coupling mechanisms for Orai and TRPC are different.142 STIM2 has not been studied as extensively as STIM1 and not at all in mast cells. The STIM isoforms differ markedly in their rates of oligomerization and dissociation which  {    # $  $ 143 The indications are that STIM2 tightly regulates basal [Ca2 ]cytt and [Ca2 ]ERR as STIM2 migrates to peripheral punctae and activates Orai1 in response to relatively small decreases in [Ca2 ]ER.129 In contrast, STIM1 comes into play with more profound store depletion as indicated in cells from mice with conditionally targeted alleles of STIM1 and STIM2.144    *  !    # ^$ ˆ@=  #  %#     STIM2 had much smaller effects. One model based on studies with ectopic STIM proteins and whole-cell dialysis views STIM2 as constitutively active under basal conditions and negatively regulated by high [Ca2 ]cyt.145 MECHANISMS OF Ca2 ENTRY (SOCE) AND EXPORT ACROSS THE PLASMA MEMBRA R NE Ca2 Entry via TRPC and Orai Proteins TRP R Cs are a subset of a superfamily of TRPs R which include TRP R C, TRPM R (melastatin), TRPV (vanilloid), TRPA (ankyrin), TRPP (polycystin) and TRPML (mucolipin).146 All seven members of the TRPC subfamily have been investigated as potential candidates for CRAC channels and certain TRPMs are thought to negatively regulate plasma membrane polarity which, as we discuss later, is essential for maintaining SOCE activity. As noted earlier, none of the TRPCs exhibit the exact characteristics of ICRAC147,148 In particular, some TRPC channels conduct Sr2 and other divalent metal ions in addition to Ca2 and have single channel conductances several orders of magnitude greater than ICRAC. All TRPCs are activated as a consequence of activation of PLC, either through store depletion or the generation of diacylglycerol.146 Formation of a functional cation channel requires assembly of four TRPCs either as homomeric or heteromeric tetramers such as TRPC1 with TRPC3, TRPC4, TRPC5, or TRPC7 and TRPC3 with TRPC6 or TRPC7.149 *#  # #   !       # $  the complex.146 Each TRP R C monomer consists of six transmembrane segments (S1-S6) with the N- and C-terminal regions located within the cell. The pore-forming loop between S5 and S6 form part of the ion channel. The TRPCs contain, in addition, several consensus binding domains in the C-terminal portion and several ankyrin repeats in the N-terminal

72

MAST CELL BIOLOGY

region. Both the C- and N- terminals contain coiled-coil domains which are thought to be essential for oligomerization.150 Like IP3Rs, TRPCs interact with accessory proteins to form signaling complexes.149 The accessory proteins are thought to regulate the cellular destination and function of the *‚\@ *# $   !!  <   !$   < and calcium signaling such as calmodulin, immunophillins, PLC, IP3R, STIM1, PMCA and SERCA.149-151 The ankyrin regions of TRPC appear to account for strategic targeting in the plasma membrane. Some TRPCs appear to be positively regulated by IP3R through interaction with an IP3R consensus binding-domain in TRPC. One concept is that TRPCs not only mediate SOCE but, in conjunction with IP3Rs, also regulate the amplitude of the calcium signal in localized regions for specialized cellular functions.149,152 The Orai proteins are atypical cation channel with four transmembrane domains.18-21,23 Three mammalian Orai   $ #!      % !     three contain a proline/arginine-rich region in the N-terminus and a putative C-terminus coiled-coiled domain.153 *# !   ˆ   <   $  #

  #       #        of a functional CRAC channel requires formation of tetramers of Orai1.154,155 Orai1 can also oligomerize with Orai2 and Orai3 to create CRAC channels each exhibiting modest differences in ion-selectivity and inhibition by elevated [Ca2 ]cyt.156 These differences could  !  {"  # $  $  $ # # relevance has not been demonstrated. All three Orai proteins can be activated by STIM2 as well as by STIM1.145 Orai1 along with STIM1 were recognized as the elusive components underlying ICRAC through several lines of evidence.16-20 Most dramatic was the demonstration that a mutation of Orai1 % <   !   $   ˜@™ # %  %#   @2 ^{$"  *  *#   $     "   wild type Orai1.18 Overexpression of Orai1 and STIM1 together, but not individually, in a variety of cells resulted in substantial ICRAC.21-23 *#   !!   # reader should consult recent reviews on the subject,148,157-160 some focused exclusively on immunological cells,161-164 for more detailed information. We have already noted that STIM1 and Orai1 are absolutely essential for activation of mast cells in vitro and in vivo.127,128 Nevertheless, the properties of ICRAC and Orai proteins would not account for the permeability of mast cells to divalent metal ions and Ca2 or the nonselective cation currents (I (ISOC) that have been recorded in activated RBL-2H3 cells.165,166 The explanation, we believe, is the interaction of less selective TRPC channels with Orai1 and STIM1. As noted earlier, knockdown of endogenous TRPC5, STIM1, or Orai1 individually with #  ‚~ $ $ {$"  @2 as well as degranulation in RBL-2H3 cells.49  !  ! "   ˆ  %# *    $! {$"  Ca2 but not of Sr2 whereas overexpression of TRPC5 with STIM1 promotes constitutive {$"  #   2 is used here only as a diagnostic probe as it is physiologically irrelevant. The data suggest that Sr2 -permeable TRPC5 acts coordinately with Orai1 and STIM1 to allow Sr2 to permeate and induce degranulation (Fig. 2).49 In support of the view that TRPCs participate in SOCE, studies in various types of cells have demonstrated that Orai1 forms complexes with some TRPCs and in conjunction with STIM1 creates currents (I (ISOC) with properties distinct from ICRAC.49,167-169 Such interactions include TRPC1 or TRPC5 with Orai149,51,167,169 and TRPC3 or TRPC6 with Orai1, Orai2, or Orai3.168 Apart from electrophysiological recordings and ablation of SOCE currents by use of siRN R A technology, there are no pharmacological diagnostic probes. Like ICRAC,

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

73

Figure 2. Antigen-induced entry of Sr2 and Ca2 via a STIM1/Orai1/TRPC complex. Changes in cytosolic Ca2 and Sr2 were monitored by single cell imaging of fura 2-loaded RBL-2H3 cells. Cells were stimulated with antigen (Ag) in Ca2 -free medium except for the periods indicated by the bars when either 3 mM Sr2 or 1 mM Ca2 %  $  # $ *#   {   @2 from intracellular stores (1st peak), entry of Sr2 (2nd peak) and of Ca2 (3rd peak). The model proposed is that on depletion of ER stores and dissociation of Ca2 from STIM1, STIM1 ologomerizes, translocates to ER/plasma membrane junctions and interacts with a TRPC5 and Orai1 complex to activate entry of Sr2 (via TRPC5) and Ca2 (via TRPC5 and Orai1).49 Similar results have been obtained in BMMC and human mast cells derived from peripheral blood.

ISOC is blocked by low concentrations (1 +M) of La3 and 2-aminoethoxydiphenyl borate (2-APB).51 If Orai proteins interact not only among themselves but also with TRPCs this %$  $ # {"  # $ ‰ <Š #  !   *# $$  challenge, nevertheless, is to demonstrate these various combinatorial arrangements $ $    $ Polarity of Plasma Membrane Regulates Ca2 -Entry Although Ca2 ^{$"      !  # ##      Ca2 across the plasma membrane (about 1/10,000), it is also dependent on maintenance of the electrochemical polarity of the plasma membrane.170 Depolarization of the plasma membrane with high external K abolishes Ca2 ^{$"   $32 The mechanisms for setting basal membrane potential may vary among subpopulations of mast cells, but the mechanism for repolarization appears similar. Resting RBL-2H3 cells express an inwardly rectifying K channel, Kir2.1, which sets the membrane potential at about -80

74

MAST CELL BIOLOGY

mV171 whereas resting primary human cell lines have a membrane potential of about 0 mV with no demonstrable Kir current.172  #    } % {$"  Ca2 and then repolarize by a mechanism attributed to K {$" # $# # @2 -activated iKCa3.1 (also known as iKCa1) potassium channel (Fig. 1).172-174 Blockade of iKCa3.1 impairs degranulation174 and chemotaxis.175 The presence of stem cell factor (SCF), interleukin (IL)-6, or IL-10 increases the expression of functional iKCa3.1 in human mast cells and the authors suggest that this could account for the enhanced reactivity of these cells to antigen.174 Chloride channels have also been implicated in regulating membrane polarity, Ca2 ^{$"   $    *#  $  # #    !    %    #          $ of this topic in a recent review.172 The nonselective Ca2 -activated cation channels, TRPM4 and TRPM5, are thought to be negatively regulate SOCE.176 †#   {$"  ! $  ! cations in mast cells.177 *‚\ ’   $  #         depolarization and limiting Ca2 {$" ˜ ™  !  †

@  !    !! 178,179 This is evident from increased Fc¡RI-mediated Ca2 ^    {  mediators and chemotaxis in TRPM4
REGULATORS OF Ca2 SIGNALING IN MAST CELLS

75

salivary gland cells.186,187 Microarray data suggest that human umbilical cord blood-derived mast cells express PMCA1 and PMCA4188 but, in general, the functions of individual \ @      %   $  <<$  $ # \ @  a regulator of cellular calcium in most or all cells whereas PMCA2 and PMCA4 have } ^  $182 The Na /Ca2 exchangers not only enable rapid Ca2 extrusion but may also decouple SOCE from the inhibitory effects of elevated [Ca2 ]cyt. These exchangers are categorized as K -independent (designated as NCX) or K -dependent (designated as NCKX). K 189 Although both families of exchangers are bidirectional and are driven by the electrochemical Na

and Ca2 gradients across cell membranes, NCKX K family members are also dependent on the K gradient.189 Under physiologic conditions the Na /Ca2 exchangers extrude Ca2 in exchange for Na when [Ca2 ]cytt is elevated but ion exchange can be reversed by low concentrations of external Na . At normal external concentrations of Na , the "#  ! $ {$"  @2 or Sr2 , increase in [Ca2 ]cytt and degranulation in mast cells by minimizing Ca2 -dependent inhibition of SOCE channels.190-192 At low concentrations of external Na , Ca2 {$"  $ $191 and Na /Ca2 {$" appear to be closely correlated when external concentrations of Na are varied.190 This correlation is one indication of the prominence of Na /Ca2 exchange during mast cell activation. Whole cell patch-clamp recordings on RBL-2H3 cells and BMMC indicate that both K -dependent and K -independent Na /Ca2 "#  ˜   ~@Œ NCKX1 K and NCX3) operate during SOCE and that they may account for as much as 50% of the Ca2 extruded from cells when [Ca2 ]cytt is elevated.192 Another indication is that the calculated maximal rate of Ca2 {$"  _€ +M/s for Na /Ca2 exchange versus 17.5 +M/s for PMCA in stimulated RBL-2H3 cells.58 In other types of cells Na /Ca2

exchangers operate in conjunction with PMCA and SERCA to regulate [Ca2 ]cyt192 and, like PMCA and SERCA, they interact with numerous regulators.189 These include lipids, especially phosphatidylinositol 4,5-bisphosphate, as well as cytoskeletal, scaffolding and signaling proteins.

MITOCHONDRIA MODULATE CALCIUM SIGNALING The dynamics of the calcium signal are also dependent on mitochondrial activity in all cells193,194 including mast cells.37,38 Three dimensional reconstruction of electron microscopic images of RBL-2H3 cells depict mitochondria as frequently surrounded by ER58 to form ER/mitochondrial R junctions195 with access to ER R SERCA pumps.196 This 2

architecture facilitates rapid uptake of newly released Ca into mitochondria.58 Indeed, IP3 induces incremental and coordinated Ca2 -release from ER R and Ca2 -uptake into 196 mitochondria in permeabized RBL-2H3 cells. In intact RBL-2H3 cells, transfer of Ca2

 #      $  @2 oscillations where the peak [Ca2 ]cytt appears to activate a mitochondrial Ca2 uniporter.197 This uptake enables rapid depletion of ER       }  # $     $$ 193 Although mitochondria are not as intimately associated with the plasma membrane as ER R in RBL-2H3 cells,58 studies in other cells suggest that mitochondria in the vicinity of CRAC channels maintain CRAC activity by minimizing localized elevations of [Ca2 ]cyt that would otherwise inhibit CRAC activity.198,199 In polarized cells, such as pancreatic   #   #    @2 waves to the apical area.200

76

MAST CELL BIOLOGY

It was initially thought that mitochondrial uptake occurred exclusively through a mitochondrial Ca2 uniporter (MCU) which operates at relatively high [Ca2 ]cytt (i.e., 1-10 +M) $ %#      $ 201 However, it is now apparent that a Ca2 /H antiporter, the leucine-zipper-EF-hand-containing transmembrane (Letm1) protein operates at much lower [Ca2 ]cytt (100 nM or lower).202 MCU uptake is driven by a negative membrane potential maintained by the respiratory chain and is activated by calmodulin at high [Ca2 ]cyt. This allows rapid uptake of Ca2 particularly in the vicinity of Ca2

channels and during calcium transients thus restricting increases in Ca2    $ domains.193,203 Letm1, in contrast, permits slow entry of Ca2 in exchange for H (1 for 1) at low [Ca2 ]cytt and its activity is dependent on the electron transport chain. Letm1 thus enables mitochondria to decode subtle changes in [Ca2 ]cytt and regulate ATP production without risk of Ca2 overload and apoptosis.194 Mitochondrial uptake and, as a consequence, SOCE are impaired in RB R L-2H3 cells by the mitochondrial respiratory chain inhibitor, antimycin A, in combination with the ATP synthase inhibitor, oligomycin.37 Total mitochondrial calcium can reach millimolar concentrations as a freely dissociable calcium phosphate (Ca3(PO4)2) complex although free Ca2 concentrations in mitochondria ([Ca2 ]m) remain at 0.5-2 +M regardless of total calcium-load.204 Egress of Ca2 is mediated by the Na /Ca2 exchanger, NCX, which permits recycling of Ca2

across the mitochondrial membrane.204 Mitochondrial Ca2 {$"  {$" # rapid equilibrium over a wide range of loading conditions to buffer external Ca2 at 0.5-1.0 +M, the so called mitochondrial “set-point” for [Ca2 ]cyt,205 which appears to be the case in RBL-2H3 cells.38 Enhanced mitochondrial NCX activity in patients with #$ #  *    $  $ #  @2 levels and function along with aberrant calcium signaling in cells from these patients.206

THE SIGNATURE OF CALCIUM SIGNALS AND FUNCTIONAL CONSEQUENCES IN MAST CELLS Calcium signals in individual mast cells are generally apparent as a sustained increases or oscillations in [Ca2 ]cyt. In RBL-2H3 cells, oscillations are of variable amplitude and duration and are usually superimposed on a rising then decreasing baseline of elevated [Ca2 ]cyt.33 The oscillations are not dependent on transient changes in membrane potential nor are they induced by ionomycin33 or thapsigargin.38 In both RBL-2H3 cells and BMMC, the oscillations are preceded by a Ca2 wave that often originates in cell protrusions.50 Ca2 -entry is required to sustain oscillations beyond the initial few oscillations.33 Localized application of immobilized antigen results in nearby repetitive Ca2 puffs that are generated by Ca2 release from stores without stimulating Ca2 entry or formation of propagating waves.50 These results, in the context of studies in other types of cells,207,208 suggest that initial Ca2 puffs result from IP3-induced release of Ca2 from ER R which then evolve into waves and regenerative oscillations on widespread replenishment of ER R stores by SOCE. *# $$  #       #% $$  $#   cells,209,210 encode calcium signals to promote a particular response or subset of responses. We have already noted that mast cell degranulation14, formation of the eicosanoid precursor arachidonic acid,15,211 chemotaxis,11 and production of at least some cytokines212-214 are dependent on SOCE. Yet under certain conditions mast cells can be stimulated to undergo chemotaxis11,215,216 or produce cytokines4 without degranulation. Preferential release of either histamine or eicosanoids has been noted with different stimulants80,217,218 and

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

77

attributed to differences in the amplitude and duration of the calcium signal.218 Although    $   #        $  calcium signals, as is true for other types of cells. The explanation as to how mast cells        $  #" %#$  {  mediators may lie in part on the nature of costimulatory signals but responses that require extensive cell remodeling such as degranulation or chemotaxis may still require calcium  %#     $  ="   $     %## $#  #  &#  <% is that transient Ca2 oscillations generated by release of Ca2 from intracellular stores %#$ {$"   $  $   $  ^$ ‚†ƒ^_`Œ 219 However, sustained Ca2 oscillations or global increases of [Ca2 ]cyt $   {$" of external Ca2 are required for degranulation.13,15,220 A similar tight dependency on SOCE was noted for the recruitment of protein kinase C and components of the ERK/ K PLA2/5-lipoxygenase pathway in RBL cells activated with thapsigargin15 or receptor agonists.220 Stimulants such as agonists of the adenosine A3221 and purinergic P2Y220 receptors elicit transient production of IP3 and release of stored Ca2 without activating $ {$"   $  $  !  # @2 -entry and activation of ICRAC is an all-or-none reaction in individual cells regardless of concentration of stimulant.220 The authors conclude that the typical concentration-response curves for  $  {          # # variation in strength of signal within individual cells.

PHARMACOLOGICAL PROBES: SOCE INHIBITORS EMERGE AS POTENTIAL THERA R PEUTIC AGENTS Pharmacological probes that have been traditionally used to investigate calcium signaling include IP 3R activators (adenophostins) and inhibitors (heparin and xestospongins), SERCA inhibitors (thapsigargin and cyclopiazonic acid) and SOCE inhibitors (2-aminoethoxydiphenyl borate (2-APB)) although none of these have therapeutic application. Recently SOCE or CRAC channel inhibitors have emerged as potential # $   #       # {  $ 222 *#   #    ! %  $  #    !%1 and they are discussed here only so far as their actions pertain to mast cells. The CRAC/ SOCE inhibitors are discussed in more detail because of their therapeutic potential. Traditional Probes for Investigation pf IP3* $*  ($  The adenophostins (A and B) and their synthetic derivatives223 are the most potent IP3R agonists described to date but, because they are cell impermeant and diffuse slowly in cytosol, their administration require patch-clamp techniques. Adenophostin A has   #$ ^      \3R than IP3224 and induces quantal release of Ca2 ,225 intracellular Ca2 oscillations and activation of ICRAC.226 However, these actions are restricted to regions where adenophostin remains localized to suggest that Ca2

oscillations and entry do not extend into regions beyond the range of activated IP3Rs.227 In RBL1 cells, adenophostin A can induce Ca2 oscillations when Ca2 entry into the cell is blocked by voltage clamp to indicate that the oscillations are regenerative that is, reuptake of Ca2  =‚ #   $   #    

78

MAST CELL BIOLOGY

reuptake.226 Activation of ICRAC by adenophostin A, as is true for thapsigargin or IP3, is substantially diminished by mitochondrial depolarization.199 There are no ideal inhibitors of IP3R-mediated Ca2 release. The membrane-permeant macrocyclic xestospongins (B, C and D) from sponge inhibit IP3-elicited Ca2 -release without affecting IP3 binding228 but they have slow action and may inhibit SERCA in some types of cells.229,230 In RBL-2H3 cells, xestospongin C (3–10 +M) inhibits intracellular   {$"  @2 , depletion of intracellular stores by IP3 and degranulation.231 However, activation of SOCE after store-depletion with thapsigargin is unaffected. The widely used SOCE activator, thapsigargin, was shown to be a mast cell activator232-234 before it was found to deplete ER Ca2 -stores by blocking SERCA-mediated reuptake of Ca2 into these stores.47 In the absence of external Ca2 , thapsigargin induces a slow increase and then decrease in [Ca2 ]cytt as stores become depleted. As is the case with antigen-stimulated Ca2 -deprived cells (Fig. 2), subsequent provision of Sr2 or Ca2

$ $ {$"  #   ‚†ƒ^_`Œ   †

@ '  #   and siRN R A technology demonstrated that TRPC5 conducted both ions whereas Orai1 conducted only Ca2 and that both channel proteins where activated by STIM1 following stimulation with either antigen or thapsigargin.49 Thapsigargin inhibits SERCA in a stoichiometric manner by tightly binding to a single site that is common to the SERCAs but not PMCAs.43,235 *#  # !  ##     #  =‚@  236 such that its action is virtually irreversible.43,237 The mycotoxin cyclopiazonic acid, in contrast, is a reversible and a less potent inhibitor of SERCA but, like thapsigargin, it suppresses SERCA Ca2 -ATPase activity. The net effects of thapsigargin and cyclopiazonic acid on RBL-2H3 cells and BMMC are similar in that both activate a SOCE pathway that is permeable to Ca2 and other divalent metal ions38,48 which is impaired by microtubular disrupting agents.238 This impairment was attributed to the inability of ER R to communicate with SOCE channels in the plasma membrane or, reinterpreted in the light of current knowledge, to disrupted communication between STIM1 in ER R and Orai or TRPCs in the plasma membrane. _^\† %     # \3-induced Ca2 -release from stores without affecting IP3 binding but later studies indicated that it blocked IP3R-mediated SOCE239 as well.1,240 2-APB has a biphasic action in RBL1 cells and other types of cells.241 ICRAC is enhanced and then suppressed at, respectively, low (1-5 +M) and high (10 +M or greater) concentrations of 2-APB. Similar biphasic actions have been noted with 2-APB on overexpressed Orai proteins.242-245 2-APB also suppresses uptake and “leakage” of Ca2 from ER R pools and release from mitochondria but the compound can be a useful reagent “when used cautiously”.246 %" +8  *   * ($  The importance of SOCE in regulating the immunological responses of mast cells, #  # {   # $  # !  ! inhibitors of SOCE as potential therapeutic agents for the treatment of allergic and autoimmune diseases. Much of the research has been conducted by the pharmaceutical industry using high-throughput screening procedures. One approach is based on {$ ^ $   ¥@2 ]cytt following depletion of intracellular stores by receptor agonists linked to IP3 production, thapsigargin, or cyclopiazonic acid. Another is based on whole cell patch-clamp technology which permits more precise #  }  ˆ@= #  # {$  #  #   $#

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

79

between ICRAC (i.e.,Orai) and TRPC channel activity and can exclude the possibility that the inhibitors block SOCE activity by inducing membrane depolarization.222 These distinctions have not always been recognized and the term “CRAC inhibitor” should be $ "$!  #     $#  #    # characteristics and the term “SOCE inhibitor” when this is not the case. Either type of inhibitor, however, should be equally interesting in terms of therapeutic potential (for an informative overview see ref. 222). More selective compounds than 2-APB were discovered after a systematic investigation of 6,5-heterocyclic compounds by investigators at the Abbott and Astellas Pharma (formerly Yamanouchi Pharmaceuticals) laboratories. Ultimately a series of highly  Œ^˜ {$ #™ }  !! %  ! # $  ICRAC. One such compound, BTP2 (otherwise known as YM58483 and now available commercially), selectively blocked thapsigargin-induced SOCE in Jurkat T-lymphocytes (IC50  150 nM).247 as well as IP3-evoked ICRAC in Jurkat and RBL-2H3 cells (IC50  2.2 and 0.5 +M, respectively).180 Interestingly, BTP2 also activates the membrane-depolarizing channel, TRPM4, at nanomolar concentrations and this action probably accounts for the potent BTP2 inhibitory effects on SOCE (as opposed to ICRAC) and the associated production of IL-2 in Jurkat cells.180 Unfortunately, similar comparisons were not reported for RBL-2H3 cells as BTP2 would appear to be useful in distinguishing between the contributions of CRAC and TRPM4 channels on SOCE and functional responses in mast cells. BTP2 also inhibits store-dependent Ca2 and Sr2 {$" # $# "  *‚\@Œ and TRPC5.248 Structurally similar inhibitors of SOCE and of IL-2 production in T cells have been reported by Boehringer Ingelheim in a series of patent applications.(see ref. 222 for patent citations). BTP2 has proved effective in such models as mouse contact hypersensitivity,247 antigen-induced eosinophilia in rodent asthma models, mouse graft versus host disease249,250 and sheep red blood cell-induced delayed hypersensitivity in mouse. However, little is reported about its side-effects. Other CRAC channel inhibitors have emerged from studies at Synta Pharmaceuticals. Of these, Synta 66 (GSK1349571A) was reported to be a relatively potent (IC50  1.4 +M) inhibitor of ICRAC in RBL-2H3 cells and thapsigargin-induced Ca2 {$" ˜ˆ@=™  Jurkat T cells without affecting TRPM4 and other channel functions.222 Synta 66 also caused a modest reduction of T-cell proliferation and concentration-dependent reductions in expression of T-bet, IL-2 and IL-17 in vitro in biopsy specimens from humans with {  % 251 #$# # $#        !$ #      of the CRAC inhibitors, the number of patent applications indicate considerable interest  @‚@ #   #    {  222 *#   of potent, selective inhibitors of these ion channels should be facilitated by the recent discovery of ORAI1 and STIM1 and new high-throughput ion channel inhibition assays.252 However, the current emphasis on CRAC/Orai inhibitors should not exclude TRPCs and TRPM4 as potential targets for reasons discussed throughout this chapter. Therefore, # {$    #  # #$  $ $ @  the current search for new channel inhibitors might provide new opportunities for the treatment of mast cell-related diseases. Their inherent advantage would be their rapid action because of the immediacy of the calcium signal during the activation of mast cells. Nevertheless, undesired effects will always remain a paramount issue because of the ubiquity of SOCE in cell signaling and here targeted drug disposition would be crucial in developing effective drug therapy.

80

MAST CELL BIOLOGY

DISEASE CORRELATES No mast cell-related disease has been directly linked to genetically-related defects in calcium signaling but other human diseases have been so linked. In addition to the presenilin mutations associated with Alzheimer’s disease, other examples include loss-of function mutations of Orai118,253 and STIM1254 in SCID, inactivating mutations of SERCA1in Brody’s disease of muscle,182 gain-off function mutation of SERCA2 in Darier’s skin disease,255 an inactivating mutation in Golgi SPCA1 in Hailey-Hailey skin disease,203 and spontaneous deafness-inducing PMCA mutations.182 While none of these mutations have been directly linked to atopic disease in humans, it has been inferred that #   ˆ   *     # $   $$ diseases254 and be expected to ameliorate allergic disease on the basis of studies in experimental animal models.127,128 In addition to the above, gain-off function mutations in TRPM4 and TRPC6 6 results in familial heart block type256 and glomerulosclerosis,257,258 respectively. As noted previously, TRPM4 is implicated in the regulation of SOCE, degranulation and migration of mast cells178,179 but the impact of mutant TRPM4 on mast cell function has not been determined. TRPC6 is expressed in mast cells49 but its function there is unknown. Numerous diseases have been mapped to chromosomal loci containing TRP channel genes but await demonstration of direct linkages.259,260 Given the variety of genetically-related defects in calcium signaling in humans and the central role of calcium signaling in the activation     #  !!  {  $     % #%#  $ $ #  # $#   $    {  disease in some subsets of patients.

CONCLUSION Primary and cultured mast cell lines have been widely used for studies of calcium signaling over the past 40 years and we have learnt much about SOCE-dependent signaling from studies of these cells. Indeed, the mast cell will remain an ideal model  $$   #  # "   $      responses. If so, we need to understand how the activities of the various Ca2 channels and sensors in different organelles are orchestrated to produce the appropriate calcium signal. Evidence points to the formation of signaling complexes embedded in specialized membrane-microdomains that facilitate the migration and interactions of channels and sensors at ER/PM and ER/mitochondrial junctions. Also unknown is whether additional {"     !  #   ˆ   *‚\@      various hetero-tetrameric combinations. These are generic questions applicable to many types of cells. There has been surprisingly little activity in regard to defects in calcium signaling in atopic and autoimmune diseases even though there are now well documented examples of genetic diseases related to defects in the operation of SOCE, SERCA, TRPM4 and NCX. We suspect that this topic as well as examination of the novel SOCE/CRAC inhibitors will be key topics for future research on mast-cell related diseases.

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

81

ADDENDUM Since completion of this manuscript Srikanth et al (Nature Cell Biol 2010; 12:436-46) have described a CRAC regulator (CRACR2A) that might account for the suppression of CRAC activity by high [Ca2 ]cyt. At basal [Ca2 ]cyt CRACR2A enhances binding of STIM to Orai thereby promoting SOCE but it dissociates from the STIM/Orai complex at high [Ca2 ]cytt and inhibits SOCE. Apparently, CRACR2A and calmodulin compete for the same Orai1 binding site which is consistent with the previously described inactivation of Orai1 by calmodulin.

ACKNOWLEDGMENTS Our studies are supported by the intramural program of the National Heart, Lung and Blood Institute of the National Institutes of Health.

REFERENCES 1. Ma HT, Beaven MA. Regulation of Ca2 signaling with particular focus on mast cells. Crit Rev Immunol 2009; 29:155-86. 2. Hoth M, Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 1992; 355:353-6. 3. Hoth M, Penner R. Calcium release-activated calcium current in rat mast cells. J Physiol (Lond) 1993; 465:359-86. ’ `$ *‚     *<}< @     ¡RI mediate unique and convergent signals for release  {      #$   † _••’– •’_’•^” 5. Ali H, Cunha-Melo JR, Saul WF et al. The activation of phospholipase C via adenosine receptors provides synergistic signals for secretion in antigen stimulated RBL-2H3 cells: Evidence for a novel adenosine receptor. J Biol Chem 1990; 265:745-53. 6. Feoktistov I, Biaggioni I. Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells. An enprofylline-sensitive mechanism with implications for asthma. J Clin Invest 1995; 96:1979-86. 7. Linden J, Thai T, Figler H et al. Characterization of human A2B adenosine receptors: radioligand binding, western blotting and coupling to Gq in human embryonic kidney 293 cells and HMC-1 mast cells. Mol Pharmacol 1999; 56:705-13. 8. Nguyen M, Solle M, Audoly LP et al. Receptors and signaling mechanisms required for prostaglandin E2-mediated regulation of mast cell degranulation and IL-6 production. J Immunol 2002; 169:4586-93. 9. Kuehn K HS, Beaven MA, Ma HT et al. Synergistic activation of phospholipases Ca and C`: a novel mechanism for PI3K-independent enhancement of Fc¡RI-induced mast cell mediator release. Cell Signal 2008; 20:625-36. 10. Ali H, Sozzani S, Fisher I et al. Differential regulation of formyl peptide and platelet-activating factor receptors. Role of phospholipase C` phosphorylation by protein kinase A. J Biol Chem 1998; 273:11012-6. 11. Hartmann K, Henz BM, Kruger K -Krasagakes K S et al. C3a and C5a stimulate chemotaxis of human mast cells. Blood 1997; 89:2863-70. 12. Qiao H, Andrade MV, Lisboa FA et al. Fc¡RI and Toll-like receptors mediate synergistic signals to markedly $  $  {  <  $    † _••“– •”“•^— 13. Beaven MA, Rogers J, Moore JP et al. The mechanism of the calcium signal and correlation with histamine release in 2H3 cells. J Biol Chem 1984; 259:7129-36. 14. Ozawa K, Szallasi Z, Kazanietz MG et al. Ca2 -dependent and Ca2 -independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells: Reconstitution of secretory responses with Ca2  $   }  %#  }  > † @# €€Œ– _“—”’€^“ 15. Chang WC, Nelson C, Parekh AB. Ca2 {$" # $# @‚@ # !  ## A2, leukotriene C4 secretion and expression of c-fos through ERK-dependent and -independent pathways in mast cells. FASEB J 2006; 20:2381-3. 16. Liou J, Kim ML, Heo WD et al. STIM is a Ca2 sensor essential for Ca2 -store-depletion-triggered Ca2

{$" @$ † _••– _Œ^’

82

MAST CELL BIOLOGY

17. Roos J, DiGregorio PJ, Yeromin AV et al. STIM1, an essential and conserved component of store-operated Ca2 channel function. J Cell Biol 2005; 169:435-45. — <  %< š \ <     $  ˆ  $ $      @‚@ channel function. Nature 2006; 441:179-85. 19. Vig M, Peinelt C, Beck A et al. CRACM1 is a plasma membrane protein essential for store-operated Ca2

entry. Science 2006; 312:1220-3. 20. Zhang SL, Yeromin AV, Zhang XH et al. Genome-wide RN R Ai screen of Ca2 {$"    # regulate Ca2 release-activated Ca2 channel activity. Proc Natl Acad Sci USA 2006; 103:9357-62. 21. Soboloff J, Spassova MA, Tang XD et al. Orai1 and STIM reconstitute store-operated calcium channel function. J Biol Chem 2006; 281:20661-5. 22. Mercer JC, Dehaven WI, Smyth JT et al. Large store-operated calcium selective currents due to co-expression of Orai1 or Orai2 with the intracellular calcium sensor, Stim1. J Biol Chem 2006; 281:24979-90. _Œ \ @ ž   ƒ      @‚@ $   *   @‚@  ˜ˆ ™ Nat Cell Biol 2006; 8:771-3. 24. Lichtenstein LM, Osler AG. Studies on the mechanism of hypersensitiviy phenomena: IX. Histamine release from human leukocytes by ragweed pollen. J Exp Med 1964; 120:507-30. 25. Greaves MW, Mongar JL. The mechanism of anaphylactic histamine release from rabbit leucocytes. Immunology 1968; 15:743-9. 26. Foreman JC, Mongar JL. The role of the alkaline earth ions in anaphylactic histamine secretion. J Physiol (Lond) 1972; 224:753-69. 27. Riley JF. The Mast Cells. Edinburgh and London: E. and S. Livingstone; 1959. 28. Foreman JC, Hallett MB, Mongar JL. Movement of strontium ions into mast cells and its relationship to the secretory response. J Physiol (Lond) 1977; 271:233-51. 29. Foreman JC, Hallet MB, Mongar JL. The relationship between histamine secretion and 45calcium-uptake by mast cells. J Physiol 1977; 271:193-214. 30. Foreman JC, Mongar JL. The action of lanthanum and manganese on anaphylactic histamine secretion. Br J Pharmacol 1973; 48:527-37. 31. Pearce FL, White JR. Effect of lanthanide ions on histamine secretion from rat peritoneal mast cells. Brr J Pharmacol 1981; 72:341-7. 32. Mohr FC, Fewtrell C. Depolarization of rat basophilic leukemia cells inhibits calcium uptake and exocytosis. J Cell Biol 1987; 104:783-92. 33. Millard PJ, Ryan R TA, Webb WW et al. Immunoglobulin E receptor cross-linking induces oscillations in intracellular free ionized calcium in individual tumor mast cells. J Biol Chem 1989; 264:19730-9. 34. Jones SV, Choi OH, Beaven MA. Carbachol induces secretion in a mast cell line (RBL-2H3) transfected with the m1 muscarinic receptor gene. FEBS Lett 1991; 289:47-50. 35. Marcotte GV, Millard PJ, Fewtrell C. Release of calcium from intracellular stores in rat basophilic leukemia    %# # {$    #   > @ \# €€•– ’_”—^—— Œ“ `  †!  @$ {$"      ˜‚†ƒ^_`Œ™  '  $!       #      " > † @# €€– _““__^€ 37. Mohr FC, Fewtrell C. The effect of mitochondrial inhibitors on calcium homeostasis in tumor mast cells. Am J Physiol 1990; 258:C217-C226. Œ—  `   ^=  ‚     #    {$"  @2 in rat basophilic ‚†ƒ^_`Œ     # # %    ’^ ##^! and mitochondrial Ca2 stores. Biochem J 1994; 304:431-40. 39. Mohr FC, Fewtrell C. The relative contributions of extracellular and intracellular calcium to secretion from tumor mast cells. Multiple effects of the proton ionophore carbonyl cyanide m-chlorophenylhydrazone. J Biol Chem 1987; 262:10638-43. 40. Streb H, Bayerd:orffer E, Haase W et al. Effect of inositol-1,4,5-trisphosphate on isolated subcellular fractions of rat pancreas. J Membr Biol 1984; 81:241-53. 41. Furuichi T, Yoshikawa S, Miyawaki A et al. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature 1989; 342:32-8. 42. Mignery GA, Newton CL, Archer BT III et al. Structure and expression of the rat inositol 1,4,5-trisphosphate receptor. J Biol Chem 1990; 265:12679-85. 43. Lytton J, Westlin M, Hanley MR. Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps. J Biol Chem 1991; 266:17067-71. 44. Thastrup O, Cullen PJ, Drbak BK K et al. Thapsigargin, a tumor promoter, discharges intracellular Ca2 stores by   #  #  $$ @2 -ATPase. Proc Natl Acad Sci USA 1990; 87:2466-70. 45. Takemura H, Hughes AR, Thastrup O et al. Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells. Evidence that an intracellular calcium pool and not an inositol phosphate regulates $ {$"  #    > † @# €—€– _“’__““^”

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

83

46. Jackson TR, Patterson SI, Thastrup O et al. A novel tumor promoter, thapsigargin, transiently increases cytoplasmic free Ca2 without generation of inositol phosphates in NG115- 401L neuronal cells. Biochem J 1988; 253:81-6. 47. Putney JW Jr. Recent breakthroughs in the molecular mechanism of capacitative calcium entry (with thoughts on how we got here). Cell Calcium 2007; 42:103-10. 48. Falcone D, Fewtrell C. Ca2 -ATPase inhibitor, cyclopiazonic acid, releases Ca2 from intracellular stores in RBL-2H3 mast cells and activates a Ca2 {$" #% #     $   J Cell Physiol 1995; 164:205-13. 49. Ma HT, Peng Z, Hiragun T et al. Canonical transient receptor potential 5 channel in conjunction with Orai1 and STIM1 allows Sr2    {$"  @2 and degranulation in a rat mast cell line. J Immunol 2008; 180:2233-9. 50. Cohen R, Torres A, Ma HT et al. Ca2 waves initiate antigen-stimulated Ca2+ responses in mast cells. J Immunol 2009; 183:6478-88. 51. Cheng KT, Liu X, Ong HL et al. Functional requirement for Orai1 in store-operated TRPC1-STIM1 channels. J Biol Chem 2008; 283:12935-40. 52. Liao Y, Erxleben C, Abramowitz J et al. Functional interactions among Orai1, TRPCs and STIM1 suggest a STIM-regulated heteromeric Orai/TRPC model for SOCE/Icrac channels. Proc Natl Acad Sci USA 2008; 105:2895-900. 53. Beaven MA, Moore JP, Smith GA et al. The calcium signal and phosphatidylinositol breakdown in 2H3 cells. J Biol Chem 1984; 259:7137-42. 54. Pribluda VS, Metzger H. Calcium-independent phosphoinositide breakdown in rat basophilic leukemia cells. Evidence for an early rise in inositol 1,4,5-trisphosphate which precedes the rise in other inositol phosphates and in cytoplasmic calcium. J Biol Chem 1987; 262:11449-54. 55. Park DJ, Min HK, R Rhee SG. IgE-induced tyrosine phosphorylation of phospholipase C-a1 in rat basophilic leukemia cells. J Biol Chem 1991; 266:24237-40. 56. Ali H, Fisher I, Haribabu B et al. Role of phospholipase C` phosphorylation in the desensitization of cellular responses to platelet-activating factor. J Biol Chem 1997; 272:11706-9. 57. Choi OH, Lee JH, Kassessinoff T et al. Carbachol and antigen mobilize calcium by similar mechanisms in a transfected mast cell line (RBL-2H3 cells) that expresses m1 muscarinic receptors. J Immunol 1993; 151:5586-95. 58. Mazel T, Raymond R, Raymond-Stintz M et al. Stochastic modeling of calcium in 3D geometry. Biophys J 2009; 96:1691-706. 59. Barker SA, Caldwell KK, Pfeiffer JR R et al. Wortmannin-sensitive phosphorylation, translocation and activation of PLCa1, but not PLCa2, in antigen-stimulated RBL-2H3 mast cells. Mol Biol Cell 1998; 9:483-96. 60. Saitoh S, Arudchandran R, Manetz TS et al. LAT is essential for Fc¡RI-mediated mast cell activation. Immunity 2000; 12:525-35. 61. Tkaczyk C, Beaven MA, Brachman SM et al. The phospholipase Ca1-dependent pathway of Fc¡RI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase. J Biol Chem 2003; 278:48474-84. “_     *<}< @    #%  ^ ! ~ ‚! $ _••“– 6:218-30. 63. Parravinci V, Gadina M, Kovarova M et al. Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation. Nat Immunol 2002; 3:741-8. 64. Thompson HL, Marshall CJ, Saklatvala J. Characterization of two different forms of mitogen-activated protein kinase kinase induced in polymorphonuclear leukocytes following stimulation by N-formylmethionyl-leucyl-phenylalanine or granulocyte-macrophage colony-stimulating factor. J Biol Chem 1994; 269:9486-92. “ $# `      ^$    #    ¡RI-mediated mast cell activation. Immunol Lett 2007; 113:59-69. 66. Thudichum JLW. A Treatise on the Chemical Constitution of the Brain. London: Baillière, Tindall and Cox; 1884. 67. Sourkes TL. The Life and Work of J. L. W. Thudichum, 1829-1901: "A Most Celebrated Exponent of the Art of Medicine and Chemistry". Montreal: Osler Libray, McGill University; 2003. 68. Beaven MA. Division of labor: Specialization of sphingosine kinases in mast cells. Immunity 2007; 26:271-3. 69. Meyer zu Heringdorf D. Lysophospholipid receptor-dependent and -independent calcium signaling. J Cell Biochem 2004; 92:937-48. 70. Choi OH, Kim JH, Kinet JP. Calcium mobilization via the sphingosine kinase in signalling by the Fc¡RI antigen receptor. Nature 1996; 380:634-6. 71. Melendez AJ, Khaw K AK. Dichotomy of Ca2 signals triggered by different phospholipid pathways in antigen stimulation of human mast cells. J Biol Chem 2002; 277:17255-62.

84

MAST CELL BIOLOGY

72. Olivera A, Mizugishi K, Tikhonova A et al. The sphingosine kinase-sphingosine-1-phosphate axis is a determinant of mast cell function and anaphylaxis. Immunity 2007; 26:287-97. 73. Mitra P, Oskeritzian CA, Payne SG et al. Role of ABCC1 in export of sphingosine-1-phosphate from mast cells. Proc Natl Acad Sci USA 2006; 103:16394-9. 74. Oskeritzian CA, Price MM, Hait NC et al. Essential roles of sphingosine-1-phosphate receptor 2 in human mast cell activation, anaphylaxis and pulmonary edema. J Exp Med 2010; 207:465-74. 75. Jolly PS, Bektas M, Olivera A et al. Transactivation of Sphingosine-1-Phosphate Receptors by Fc¡RI Triggering Is Required for Normal Mast Cell Degranulation and Chemotaxis. J Exp Med 2004; 199:959-70. 76. Olivera A, Urtz N, Mizugishi K et al. IgE-dependent activation of spingosine kinase 1 and 2 and secretion of sphingosine-1-phosphate requires Fyn kinase and contributes to mast cell responses. J Biol Chem 2006; 281:2515-25. 77. R Ryu SD, Lee HS, Suk HY et al. Cross-linking of Fc¡RI causes Ca2 mobilization via a sphingosine kinase pathway in a clathrin-dependent manner. Cell Calcium 2009; 45:99-108. 78. Hait NC, Oskeritzian CA, Paugh SW et al. Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases. Biochim Biophys Acta 2006; 1758:2016-26. 79. Rivera J, Proia RL, Olivera A. The alliance of sphingosine-1-phosphate and its receptors in immunity. Nat Rev Immunol 2008; 8:753-63. 80. Melendez AJ. Allergy therapy: the therapeutic potential of targeting sphingosine kinase signalling in mast cells. Eur J Immunol 2008; 38:2969-74. 81. Vermassen E, Parys JB, Mauger JP. Subcellular distribution of the inositol 1,4,5-trisphosphate receptors: functional relevance and molecular determinants. Biol Cell 2004; 96:3-17. 82. Michelangeli F, Ogunbayo OA, Wootton LL. A plethora of interacting organellar Ca2 stores. Curr Opin Cell Biol 2005; 17:135-40. 83. Devogelaere B, Verbert L, Parys JB et al. The complex regulatory function of the ligand-binding domain of the inositol 1,4,5-trisphosphate receptor. Cell Calcium 2008; 43:17-27. 84. Choe CU, Ehrlich BE. The inositol 1,4,5-trisphosphate receptor (IP3R) and its regulators: sometimes good and sometimes bad teamwork. Sci STKE 2006; 2006:re15. 85. Patterson RL, Boehning D, Snyder SH. Inositol 1,4,5-trisphosphate receptors as signal integrators. Annu Rev Biochem 2004; 73:437-65. 86. Vanlingen S, Parys JB, Missiaen L et al. Distribution of inositol 1,4,5-trisphosphate receptor isoforms, SERCA isoforms and Ca2 binding proteins in RBL-2H3 rat basophilic leukemia cells. Cell Calcium 1997; 22:475-86. 87. Wilson BS, Pfeiffer JR, Smith AJ et al. Calcium-dependent clustering of inositol 1,4,5-trisphosphate receptors. Mol Biol Cell 1998; 9:1465-78. 88. Woodard GE, Lopez JJ, Jardin I et al. TRPC3 regulates agonist-stimulated Ca2 mobilization by mediating the interaction between type I inositol 1,4,5-trisphosphate receptor, RACK1 and Orai1. J Biol Chem 2010; 285:8045-53. 89. Mikoshiba K. The IP3 receptor/Ca2 channel and its cellular function. Biochem Soc Symp 2007:9-22. 90. Tu H, Wang Z, Bezprozvanny I. Modulation of mammalian inositol 1,4,5-trisphosphate receptor isoforms by calcium: a role of calcium sensor region. Biophys J 2005; 88:1056-69. 91. Vanderheyden V, Devogelaere B, Missiaen L et al. Regulation of inositol 1,4,5-trisphosphate-induced Ca2

release by reversible phosphorylation and dephosphorylation. Biochim Biophys Acta 2009; 1793:959-70. 92. Jayaraman T, Ondrias K, Ondriasova E et al. Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science 1996; 272:1492-4. 93. Yokoyama K, Su II, Tezuka T et al. BAN A K regulates BCR-induced calcium mobilization by promoting tyrosine phosphorylation of IP3 receptor. EMBO J 2002; 21:83-92. 94. Echevarria W, Leite MF, Guerra MT et al. Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum. Nat Cell Biol 2003; 5:440-6. 95. Pinton P, Pozzan T, Rizzuto R. The Golgi apparatus is an inositol 1,4,5-trisphosphate-sensitive Ca2 store, with functional properties distinct from those of the endoplasmic reticulum. EMBO J 1998; 17:5298-308. 96. Turner H, Fleig A, Stokes A et al. Discrimination of intracellular calcium store subcompartments using TRPV1 (transient receptor potential channel, vanilloid subfamily member 1) release channel activity. Biochem J 2003; 371:341-50. 97. Huang Y, Putney JW Jr. Relationship between intracellular calcium store depletion and calcium release-activated calcium current in a mast cell line (RBL-1). J Biol Chem 1998; 273:19554-9. 98. Castro J, Aromataris EC, R Rychkov GY et al. A small component of the endoplasmic reticulum is required for store-operated Ca2 channel activation in liver cells: evidence from studies using TRPV1 and taurodeoxycholic acid. Biochem J 2009; 418:553-66. 99. Ong HL, Liu X, Tsaneva-Atanasova K et al. Relocalization of STIM1 for activation of store-operated Ca2

entry is determined by the depletion of subplasma membrane endoplasmic reticulum Ca2 store. J Biol Chem 2007; 282:12176-85.

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

85

100. Bola B, Allan V. How and why does the endoplasmic reticulum move? Biochem Soc Trans 2009; 37:961-5. 101. Smyth JT, Dehaven WI, Bird GS et al. Role of the microtubule cytoskeleton in the function of the store-operated Ca2 channel activator STIM1. J Cell Sci 2007; 120:3762-71. 102. Wu S, Chen H, Alexeyev MF et al. Microtubule motors regulate ISOC activation necessary to increase endothelial cell permeability. J Biol Chem 2007; 282:34801-8. 103. Means S, Smith AJ, Shepherd J et al. Reaction diffusion modeling of calcium dynamics with realistic ER geometry. Biophys J 2006; 91:537-57. 104. Wuytack F, Papp B, Verboomen H et al. A sarco/endoplasmic reticulum Ca2 -ATPase 3-type Ca2 pump is expressed in platelets, in lymphoid cells and in mast cells. J Biol Chem 1994; 269:1410-6. 105. Jousset H, Frieden M, Demaurex N. STIM1 knockdown reveals that store-operated Ca2 channels located close to sarco/endoplasmic Ca2 *\ ˜=‚@™ $    #  $$ J Biol Chem 2007; 282:11456-64. 106. Camacho P, Lechleiter JD. Increased frequency of calcium waves in Xenopus laevis oocytes that express a calcium-ATPase. Science 1993; 260:226-9. 107. Yu R, Hinkle PM. Rapid turnover of calcium in the endoplasmic reticulum during signaling. Studies with cameleon calcium indicators. J Biol Chem 2000; 275:23648-53. 108. Roderick HL, Lechleiter JD, Camacho P. Cytosolic phosphorylation of calnexin controls intracellular Ca2 oscillations via an interaction with SERCA2b. J Cell Biol 2000; 149:1235-48. 109. Lopéz JJ, Jardin I, Bobe R et al. STIM1 regulates acidic Ca2        %# =‚@Œ in human platelets. Biochem Pharmacol 2008; 75:2157-64. • ž ƒ ˆ@@ *#  ^  $ {$" " @ @$ _••–    111. Green K KN, Demuro A, Akbari Y et al. SERCA pump activity is physiologically regulated by presenilin and regulates amyloid ` production. J Cell Biol 2008; 181:1107-16. 112. Li Y, Camacho P. Ca2 -dependent redox modulation of SERCA 2b by ERp57. R J Cell Biol 2004; 164:35-46. Œ ˆ > ƒ$%< ‚   ~  !         #  ##  proteins in activated mast cells: implications for regulation of protein synthesis in the rat basophilic leukemia mast cell line RBL-2H3. J Proteome Res 2009; 8:3068-77. 114. Tu H, Nelson O, Bezprozvanny A et al. Presenilins form ER Ca2 leak channels, a function disrupted by  }# ¤ ^< $ @ _••“– _“€—^€Œ  ~ ˆ *$ ` ƒ *    }# ^< $    $ @2 leak function of presenilin 1. J Clin Invest 2007; 117:1230-9. 116. Ong HL, Liu X, Sharma A et al. Intracellular Ca2 release via the ER R translocon activates store-operated $   \{$   # _••”– ’Œ”€”^—•— 117. Van Coppenolle F, Vanden Abeele F, Slomianny C et al. Ribosome-translocon complex mediates calcium leakage from endoplasmic reticulum stores. J Cell Sci 2004; 117:4135-42. —   ƒ > ˆ¤‚ >   *  $   @2 leak in proliferating vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 2009; 296:H910-H916. 119. Vanden Abeele F, Bidaux G, Gordienko D et al. Functional implications of calcium permeability of the channel formed by pannexin 1. J Cell Biol 2006; 174:535-46. 120. Szewczyk MM, Pande J, Grover AK. Caloxins: a novel class of selective plasma membrane Ca2 pump #   $ # \{$   # ^=$ > \# _••—– ’“_^““ 121. Cheung KH, Shineman D, Muller M et al. Mechanism of Ca2  $  }# ¤   presenilin regulation of InsP3 receptor channel gating. Neuron 2008; 58:871-83. 122. Yoo AS, Cheng I, Chung S et al. Presenilin-mediated modulation of capacitative calcium entry. Neuron 2000; 27:561-72. 123. Savineau JP. Is the translocon a crucial player of the calcium homeostasis in vascular smooth muscle cell? Am J Physiol Heart Circ Physiol 2009; 296:H906-H907. _’ $ <  ž @  ƒ#§< ž   \! $ < !        iPLA2-pathway regulated store operated channels activation. FASEB J 2006; 20:1215-7. 125. Wonderlin WF. Constitutive, translation-independent opening of the protein-conducting channel in the  $$ \{$   # _••€– ’”€”^Œ• 126. Beecroft MD, Taylor CW. Luminal Ca2 regulates passive Ca2 {$"   #  $    hepatocytes. Biochem J 1998; 334 ( Pt 2):431-5. 127. Baba Y, Nishida K, Fujii Y et al. Essential function for the calcium sensor STIM1 in mast cell activation and anaphylactic responses. Nat Immunol 2008; 9:81-8. 128. Vig M, Dehaven WI, Bird GS et al. Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels. Nat Immunol 2008; 9:89-96. 129. Brandman O, Liou J, Park WS et al. STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2 levels. Cell 2007; 131:1327-39.

86

MAST CELL BIOLOGY

130. Stathopulos PB, Li GY, Plevin MJ et al. Stored Ca2 depletion-induced oligomerization of STIM1 via the EF-SAM region: An initiation mechanism for capacitive Ca2 entry. J Biol Chem 2006; 281:35855-62. 131. Zheng L, Stathopulos PB, Li GY et al. Biophysical characterization of the EF-hand and SAM domain containing Ca2 sensory region of STIM1 and STIM2. Biochem Biophys Res Commun 2008; 369:240-6. 132. Zhang SL, Yu Y, Roos J et al. STIM1 is a Ca2 sensor that activates CRAC channels and migrates from the Ca2 store to the plasma membrane. Nature 2005; 437:902-5. 133. Luik RM, Wu MM, Buchanan J et al. The elementary unit of store-operated Ca2 entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions. J Cell Biol 2006; 174:815-25. 134. Calloway N, Vig M, Kinet JP et al. Molecular clustering of STIM1 with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2+ stores and on electrostatic interactions. Mol Biol Cell 2008; 20:389-99. 135. Muik M, Fahrner M, Derler I et al. A cytosolic homomerization and a modulatory domain within STIM1 C-terminus determine coupling to ORAI1 channels. J Biol Chem 2009; 284:8421-6. Œ“ @% ~ `%<  †  †   ‹$  *    @_¨ {$"    %# the C-terminal acidic coiled coil of Orai1. Biochemistry 2010; 49:1067-71. 137. Li Z, Lu J, Xu P et al. Mapping the interacting domains of STIM1 and Orai1 in Ca2 release-activated Ca2 channel activation. J Biol Chem 2007; 282:29448-56. 138. Park CY, Hoover PJ, Mullins FM et al. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell 2009; 136:876-90. 139. Muik M, Frischauf I, Derler I et al. Dynamic coupling of the putative coiled-coil domain of ORAI1 with STIM1 mediates ORAI1 channel activation. J Biol Chem 2008; 283:8014-22. 140. Mathes C, Fleig A, Penner R. Calcium release-activated calcium current (ICRAC) is a direct target for sphingosine. J Biol Chem 1998; 273:25020-30. 141. Yuan JP, Zeng W, Huang GN et al. STIM1 heteromultimerizes TRPC channels to determine their function as store-operated channels. Nat Cell Biol 2007; 9:636-45. 142. Zeng W, Yuan JP, Kim MS et al. STIM1 gates TRPC channels, but not Orai1, by electrostatic interaction. Mol Cell 2008; 32:439-48. 143. Stathopulos PB, Zheng L, Ikura M. Stromal Interaction Molecule (STIM) 1 and STIM2 Calcium Sensing Regions Exhibit Distinct Unfolding and Oligomerization Kinetics. J Biol Chem 2009; 284:728-32. 144. Oh-Hora M, Yamashita M, Hogan PG et al. Dual functions for the endoplasmic reticulum calcium sensors STIM1 and STIM2 in T-cell activation and tolerance. Nat Immunol 2008; 9:432-43. 145. Parvez S, Beck A, Peinelt C et al. STIM2 protein mediates distinct store-dependent and store-independent modes of CRAC channel activation. FASEB J 2008; 22:752-61. 146. Nilius B, Owsianik G, Voets T et al. Transient receptor potential cation channels in disease. Physiol Rev 2007; 87:165-217. 147. Parekh AB, Putney JW Jr. Store-operated calcium channels. Physiol Rev 2005; 85:757-810. ’— \$ >& †   @ $    ^  $ {$" > \# _••—– 586:3055-9. ’€ $<  ˆ `ƒ ˆ }  $  *‚\@ # \{$   # _••”– 455:187-200. 150. Vazquez G, Wedel BJ, Aziz O, Trebak M, Putney JW, Jr. The mammalian TRPC cation channels. Biochim Biophys Acta 2004; 1742:21-36.  !   >š   &   \ ^     $ *‚\@ # \{$  Arch 2005; 451:116-24. 152. Putney JW Jr. Inositol lipids and TRPC channel activation. Biochem Soc Symp 2007:37-45. 153. Cahalan MD, Zhang SL, Yeromin AV et al. Molecular basis of the CRAC channel. Cell Calcium 2007; 42:133-44. 154. Mignen O, Thompson JL, Shuttleworth TJ. Orai1 subunit stoichiometry of the mammalian CRAC channel pore. J Physiol 2008; 586:419-25. 155. Ji W, Xu P, Li Z, Lu J, Liu L, Zhan Y et al. Functional stoichiometry of the unitary calcium-release-activated calcium channel. Proc Natl Acad Sci USA 2008; 105:13668-73. 156. Lis A, Peinelt C, Beck A, Parvez S, Monteilh-Zoller M, Fleig A et al. CRACM1, CRACM2 and CRACM3 are store-operated Ca2 channels with distinct functional properties. Curr Biol 2007; 17:794-800. 157. Scharenberg AM, Humphries LA, Rawlings DJ. Calcium signalling and cell-fate choice in B cells. Nat Rev Immunol 2007; 7:778-89. 158. Clapham DE. Calcium signaling. Cell 2007; 131:1047-58. 159. Frischauf I, Schindl R, Derler I et al. The STIM/Orai coupling machinery. Channels (Austin ) 2008; 2:261-8. 160. Potier M, Trebak M. New developments in the signaling mechanisms of the store-operated calcium entry #% \{$   # _••—– ’”’•^

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

87

161. Luik RM, Lewis RS. New insights into the molecular mechanisms of store-operated Ca2 signaling in T-cells. Trends Mol Med 2007; 13:103-7. 162. Feske S. Calcium signalling in lymphocyte activation and disease. Nat Rev Immunol 2007; 7:690-702. 163. Oh-Hora M, Rao A. Calcium signaling in lymphocytes. Curr Opin Immunol 2008; 20:250-8. 164. Vig M, Kinet JP. Calcium signaling in immune cells. Nature Immunol 2009; 10:21-7. 165. Fasolato C, Hoth M, Matthews G et al. Ca2 and Mn2 {$" # $#  ^ !     #    \  ~   ' €€Œ– €•Œ•“—^”_ 166. Braun FJ, Broad LM, Armstrong DL et al. Stable activation of single Ca2 release-activated Ca2 channels in divalent cation-free solutions. J Biol Chem 2001; 12; 276:1063-70. 167. Ong HL, Cheng KT, Liu X, Bandyopadhyay BC, Paria BC, Soboloff J et al. Dynamic assembly of *‚\@^* ^ˆ     "  !!   ^  $ {$" =!  similarities in store-operated and calcium release-activated calcium channel components. J Biol Chem 2007; 282:9105-16. 168. Liao Y, Erxleben C, Yildirim E et al. Orai proteins interact with TRPC channels and confer responsiveness to store depletion. Proc Natl Acad Sci USA 2007; 104:4682-7. 169. Jardin I, Lopez JJ, Salido GM et al. Orai1 mediates the interaction between STIM1 and hTRPC1 and regulates the mode of activation of hTRPC1-forming Ca2 channels. J Biol Chem 2008; 283:25296-304. 170. Mohr FC, Fewtrell C. IgE receptor-mediated depolarization of rat basophilic leukemia cells measured %# # {$    ^" > $ €—”– Œ—“’^”• 171. Wischmeyer E, Lentes KU, Karschin A. Physiological and molecular characterization of an IRK-type %     #   $$    \{$   # €€– ’_€—•€^€ 172. Bradding P. Mast cell ion channels. Chem Immunol Allergy 2005; 87:163-78. 173. Narenjkar J, Assem SK, Ganellin CR. Inhibition of the antigen-induced activation of RBL-2H3 cells by cetiedil and some of its analogues. Eur J Pharmacol 2004; 483:107-16. 174. Duffy SM, Berger P, Cruse G et al. The K channel iKCA1 potentiates Ca2 {$"   $  human lung mast cells. J Allergy Clin Immunol 2004; 114:66-72. 175. Cruse G, Duffy SM, Brightling CE et al. Functional KCA3.1 K channels are required for human lung mast cell migration. Thorax 2006; 61:880-5. 176. Harteneck C. Function and pharmacology of TRPM cation channels. Naunyn Schmiedebergs Arch Pharmacol 2005; 371:307-14. 177. Launay P, Fleig A, Perraud AL et al. TRPM4 is a Ca2 -activated nonselective cation channel mediating cell membrane depolarization. Cell 2002; 109:397-407. 178. Vennekens R, Olausson J, Meissner M, Bloch W, Mathar I, Philipp SE et al. Increased IgE-dependent mast cell activation and anaphylactic responses in mice lacking the calcium-activated nonselective cation channel TRPM4. Nat Immunol 2007; 8:312-20. 179. Shimizu T, Owsianik G, Freichel M et al. TRPM4 regulates migration of mast cells in mice. Cell Calcium 2009; 45:226-32. 180. Takezawa R, Cheng H, Beck A et al. A pyrazole derivative potently inhibits lymphocyte Ca2 {$" and cytokine production by facilitating transient receptor potential melastatin 4 channel activity. Mol Pharmacol 2006; 69:1413-20. 181. Strehler EE, Treiman M. Calcium pumps of plasma membrane and cell interior. Curr Mol Med 2004; 4:323-35. 182. Brini M. Plasma membrane Ca2 -ATPase: from a housekeeping function to a versatile signaling role. \{$   # _••€– ’”“”^“’ 183. Obara K, Miyashita N, Xu C et al. Structural role of countertransport revealed in Ca2 pump crystal structure in the absence of Ca2 . Proc Natl Acad Sci USA 2005; 102:14489-96. 184. Di Leva F, Domi T, Fedrizzi L et al. The plasma membrane Ca2 ATPase of animal cells: structure, function and regulation. Arch Biochem Biophys 2008; 476:65-74. 185. Bautista DM, Hoth M, Lewis RS. Enhancement of calcium signalling dynamics and stability by delayed modulation of the plasma-membrane calcium-ATPase in human T-cells. J Physiol 2002; 541:877-94. 186. Belan PV, Gerasimenko OV, Tepikin AV et al. Localization of Ca2 extrusion sites in pancreatic acinar cells. J Biol Chem 1996; 271:7615-9. 187. Lee MG, Xu X, Zeng W et al. Polarized expression of Ca2 pumps in pancreatic and salivary gland cells. Role in initiation and propagation of [Ca2 ]i waves. J Biol Chem 1997; 272:15771-6. —— >  * ` ‚#$# ‚   ^%  "      #$   stimulated by IgE or Fc¡‚^  !  " % <   !!  {  responses. BMC Genomics 2006; 7:210-27. 189. Lytton J. Na /Ca2 exchangers: three mammalian gene families control Ca2 transport. Biochem J 2007; 406:365-82. 190. Alfonso A, Lago J, Botana MA et al. Characterization of the Na /Ca2 exchanger on rat mast cells. Evidence for a functional role on the regulation of the cellular response. Cell Physiol Biochem 1999; 9:53-71.

88

MAST CELL BIOLOGY

Rumpel E, Pilatus U, Mayer A et al. Na -dependent Ca2 transport modulates the secretory response to 191. R the Fc¡ receptor stimulus of mast cells. Biophys J 2000; 79:2975-86. 192. Aneiros E, Philipp S, Lis A et al. Modulation of Ca2 signaling by Na /Ca2 exchangers in mast cells. J Immunol 2005; 174:119-30. 193. Romagnoli A, Aguiari P, De SD et al. Endoplasmic reticulum/mitochondria calcium cross-talk. Novartis Found Symp 2007; 287:122-31. 194. Santo-Domingo J, Demaurex N. Calcium uptake mechanisms of mitochondria. Biochim Biophys Acta 2010; doi:10.1016/j.bbabio.2010.01.005. € \# \ @ ª \ # *   Ÿ$   $       ^ reticulum to the mitochondria. J Physiol 2000; 529 Pt 3:553-64. 196. Csordás G, Hajnóczky G. Sorting of calcium signals at the junctions of endoplasmic reticulum and mitochondria. Cell Calcium 2001; 29:249-62. 197. Csordás G, Hajnóczky G. Plasticity of mitochondrial calcium signaling. J Biol Chem 2003; 278:42273-82. 198. Hoth M, Button DC, Lewis RS. Mitochondrial control of calcium-channel gating: a mechanism for sustained signaling and transcriptional activation in T-lymphocytes. Proc Natl Acad Sci USA 2000; 97:10607-12. 199. Glitsch MD, Bakowski D, Parekh AB. Store-operated Ca2 entry depends on mitochondrial Ca2 uptake. EMBO J 2002; 21:6744-54. 200. Park MK, Ashby MC, Erdemli G et al. Perinuclear, perigranular and sub-plasmalemmal mitochondria have distinct functions in the regulation of cellular calcium transport. EMBO J 2001; 20:1863-74. 201. Kirichok Y, K Krapivinsky G, Clapham DE. The mitochondrial calcium uniporter is a highly selective ion channel. Nature 2004; 427:360-4. _•_ >   # ƒ @# = ^% ‚~     ƒ   #  @2 /H

antiporter. Science 2009; 326:144-7. 203. Dolman NJ, Tepikin AV. Calcium gradients and the Golgi. Cell Calcium 2006; 40:505-12. 204. Nicholls DG. Mitochondria and calcium signaling. Cell Calcium 2005; 38:311-7. 205. Nicholls DG. The regulation of extramitochondrial free calcium ion concentration by rat liver mitochondria. Biochem J 1978; 176:463-74. 206. Visch HJ, R Rutter GA, Koopman WJ et al. Inhibition of mitochondrial Na -Ca2 exchange restores agonist-induced ATP production and Ca2 #  #$ "    > † @# _••’– 279:40328-36. 207. Tovey SC, de SP, Lipp P et al. Calcium puffs are generic InsP3-activated elementary calcium signals and are downregulated by prolonged hormonal stimulation to inhibit cellular calcium responses. J Cell Sci 2001; 114:3979-89. 208. Berridge MJ. Calcium microdomains: organization and function. Cell Calcium 2006; 40:405-12. 209. Kalesnikoff J, Galli SJ. New developments in mast cell biology. Nat Immunol 2008; 9:1215-23. 210. Beaven MA. Our perception of the mast cell from Paul Ehrlich to now. Eur J Immunol 2009; 39:11-25. 211. Hirasawa N, Santini F, Beaven MA. Activation of the mitogen-activated protein kinase/cytosolic phospholipase A2 pathway in a rat mast cell line. Indications of different pathways for release of arachidonic acid and secretory granules. J Immunol 1995; 154:5391-402. 212. Wodnar-Filipowicz A, Moroni C. Regulation of interleukin 3 mRN R A expression in mast cells occurs at the posttranscriptional level and is mediated by calcium ions. Proc Natl Acad Sci USA 1990; 87:777-81. 213. Plaut M, Pierce JH, Watson CJ et al. Mast cell lines produce lymphokines in response to cross-linkage of Fc¡RI or to calcium ionophores. Nature 1989; 339:64-7. 214. Burd PR, Rogers HW, Gordon JR R et al. Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines. J Exp Med 1989; 170:245-57. 215. Thompson HL, Burbelo PD, Yamada Y et al. Mast cells chemotax to laminin with enhancement after IgE-mediated activation. J Immunol 1989; 143:4188-92. 216. Hofstra CL, Desai PJ, Thurmond RL et al. Histamine H4 receptor mediates chemotaxis and calcium mobilization of mast cells. J Pharmacol Exp Ther 2003; 305:1212-21. 217. Benyon RC, Robinson C, Church MK. Differential release of histamine and eicosanoids from human skin mast cells activated by IgE-dependent and non-immunological stimuli. Brit J Pharmacol 1989; 97:898-904. 218. van Haaster CM, Engels W, Lemmens PJ et al. Differential release of histamine and prostaglandin D2 in rat peritoneal mast cells: roles of cytosolic calcium and protein tyrosine kinases. Biochim Biophys Acta 1995; 1265:79-88. 219. Kim TD, Eddlestone GT, Mahmoud SF et al. Correlating Ca2 responses and secretion in individual RBL-2H3 mucosal mast cells. J Biol Chem 1997; 272:31225-9. 220. Chang WC, Di CJ, Nelson C et al. All-or-none activation of CRAC channels by agonist elicits graded responses in populations of mast cells. J Immunol 2007; 179:5255-63.

REGULATORS OF Ca2 SIGNALING IN MAST CELLS

89

221. Ramkumar V, Stiles GL, Beaven MA et al. The A3R is the unique adenosine receptor which facilitates release of allergic mediators in mast cells. J Biol Chem 1993; 268:16887-90. 222. Sweeney ZK, Minatti A, Button DC et al. Small-molecule inhibitors of store-operated calcium entry. ChemMedChem 2009; 4:706-18. __Œ $ #   * $  *! @   _^\ ^  $  #   ##^    # ^^  ##    !  !$  $ modeling. J Org Chem 2008; 73:1682-92. 224. Takahashi M, Tanzawa K, Takahashi S. Adenophostins, newly discovered metabolites of Penicillium brevicompactum, act as potent agonists of the inositol 1,4,5-trisphosphate receptor. J Biol Chem 1994; 269:369-72. 225. Hirota J, Michikawa T, Miyawaki A et al. Adenophostin-mediated quantal Ca2   # $    reconstituted inositol 1,4,5-trisphosphate receptor type 1. FEBS Lett 1995; 368:248-52. 226. Huang Y, Takahashi M, Tanzawa K et al. Effect of adenophostin A on Ca2 entry and calcium release-activated calcium current (Icrac) in rat basophilic leukemia cells. J Biol Chem 1998; 273:31815-21. 227. Bird GS, Takahashi M, Tanzawa K et al. Adenophostin A induces spatially restricted calcium signaling in Xenopus laevis oocytes. J Biol Chem 1999; 274:20643-9. 228. Gafni J, Munsch JA, Lam TH et al. Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 1997; 19:723-33. 229. Castonguay A, Robitaille R. Xestospongin C is a potent inhibitor of SERCA at a vertebrate synapse. Cell Calcium 2002; 32:39-47. 230. Solovyova N, Fernyhough P, Glazner G et al. Xestospongin C empties the ER R calcium store but does not inhibit InsP3-induced Ca2 release in cultured dorsal root ganglia neurones. Cell Calcium 2002; 32:49-52. 231. Oka T, Sato K, Hori M et al. Xestospongin C, a novel blocker of IP3 receptor, attenuates the increase in cytosolic calcium level and degranulation that is induced by antigen in RBL-2H3 mast cells. Brr J Pharmacol 2002; 135:1959-66. 232. Rasmussen U, Broogger CS, Sandberg F. Thapsigargin and thapsigargicin, two new histamine liberators from Thapsia garganica L. Acta Pharm Suec 1978; 15:133-40. 233. Patkar SA, Rasmussen U, Diamant B. On the mechanism of histamine release induced by thapsigargin from Thapsia garganica L. Agents Actions 1979; 9:53-7. 234. Ali H, Christensen SB, Foreman JC et al. The ability of thapsigargin and thapsigargicin to activate cells !!  # {   † > \#  €—– —”•^_ _Œ $ @  `      $$  ‹$   # #    #   # @2

ATPase SERCA. J Biol Chem 2004; 279:17973-9. 236. Wootton LL, Michelangeli F. The effects of the phenylalanine 256 to valine mutation on the sensitivity of sarcoplasmic/endoplasmic reticulum Ca2 ATPase (SERCA) Ca2 pump isoforms 1, 2 and 3 to thapsigargin and other inhibitors. J Biol Chem 2006; 281:6970-6. 237. Sagara Y, Inesi G. Inhibition of the sarcoplasmic reticulum Ca2 transport ATPase by thapsigargin at subnanomolar concentrations. J Biol Chem 1991; 266:13503-6. 238. Oka T, Hori M, Ozaki H. Microtubule disruption suppresses allergic response through the inhibition of $ {$"  #    $ #% > $ _••– ”’’—’^€ 239. Ma HT, Patterson RL, van Rossum DB et al. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2 channels. Science 2000; 287:1647-51. 240. Bootman MD, Collins TJ, Mackenzie L et al. 2-Aminoethoxydiphenyl borate (2-APB) is a reliable blocker of store-operated Ca2 entry but an inconsistent inhibitor of InsP3-induced Ca2 release. FASEB J 2002; 16:1145-50. 241. Prakriya M, Lewis RS. Potentiation and inhibition of Ca2 release-activated Ca2 channels by 2-aminoethyldiphenyl borate (2-APB) occurs independently of IP3 receptors. J Physiol 2001; 536:3-19. 242. Zhang SL, Kozak JA, Jiang W et al. Store-dependent and -independent modes regulating Caa2 release-activated Ca2 channel activity of human Orai1 and Orai3. J Biol Chem 2008; 283:17662-71. 243. Peinelt C, Lis A, Beck A et al. 2-Aminoethoxydiphenyl borate directly facilitates and indirectly inhibits STIM1-dependent gating of CRAC channels. J Physiol 2008; 586:3061-73. 244. Dehaven WI, Smyth JT, Boyles RR R et al. Complex actions of 2-aminoethyldiphenyl borate on store-operated calcium entry. J Biol Chem 2008; 283:19265-73. 245. Schindl R, Bergsmann J, Frischauf I et al. 2-Aminoethoxydiphenyl borate alters selectivity of Orai3 channels by increasing their pore size. J Biol Chem 2008; 283:20261-7. _’“ < =   &  \  < @   %      {  immune-mediated disorders. Int J Biochem Cell Biol 2004; 36:183-8. 247. Ishikawa J, Ohga K, Yoshino T et al. A pyrazole derivative, YM-58483, potently inhibits store-operated sustained Ca2 {$"  ƒ^_  $  *^# > $ _••Œ– ”•’’’^€ 248. He LP, Hewavitharana T, Soboloff J et al. A functional link between store-operated and TRPC channels !  # Œ^˜ {$ #™ }  !! †*\_ > † @# _••– _—••€€”^••“

90

MAST CELL BIOLOGY

249. Yoshino T, Ishikawa J, Ohga K et al. YM-58483, a selective CRAC channel inhibitor, prevents antigen-induced airway eosinophilia and late phase asthmatic responses via Th2 cytokine inhibition in animal models. Eur J Pharmacol 2007; 560:225-33. 250. Ohga K, Takezawa R, Yoshino T et al. The suppressive effects of YM-58483/BTP-2, a store-operated Ca2

  <   {     !    %   !! \$ \#  Ther 2008; 21:360-9. 251. de Lumley M, Hart DJ, Cooper MA et al. A biophysical characterisation of factors controlling dimerisation and selectivity in the NF-gB and NFAT families. J Mol Biol 2004; 339:1059-75. 252. Dunlop J, Bowlby M, Peri R et al. High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology. Nat Rev Drug Discov 2008; 7:358-68. 253. Feske S, Prakriya M, Rao A et al. A severe defect in CRAC Ca2 channel activation and altered K channel   *^   $   > ="  _••– _•_“^“_ _’ \  @ @  @ \    *  $  %#     $  and autoimmunity. N Engl J Med 2009; 360:1971-80. 255. Sakuntabhai A, Ruiz R -Perez V, Carter S et al. Mutations in ATP2A2, encoding a Ca2 pump, cause Darier disease. Nat Genet 1999; 21:271-7. _“  $  #$}^†# = @  ž       #  # *‚\ ’   with human progressive familial heart block type I. J Clin Invest 2009; 119:2737-44. 257. Reiser J, Polu KR, Moller CC et al. TRPC6 is a glomerular slit diaphragm-associated channel required for normal renal function. Nat Genet 2005; 37:739-44. 258. Winn MP, Conlon PJ, Lynn KL et al. A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis. Science 2005; 308:1801-4. 259. Abramowitz J, Birnbaumer L. K Know thy neighbor: a survey of diseases and complex syndromes that map to chromosomal regions encoding TRP channels. Handb Exp Pharmacol 2007:379-408. 260. Abramowitz J, Birnbaumer L. Physiology and pathophysiology of canonical transient receptor potential channels. FASEB J 2009; 23:297-328.

CHAPTER R6 SPATIO-TEMPORA R L SIGNALING IN MAST CELLS †   &| >  ˆ!    ƒ< Department of Pathology, University of New Mexico Albuquerque, New Mexico, USA |   0  3 5  &$    

Abstract:

This chapter summarizes the evidence for localized signaling domains in mast cells  # %#   $ $  # ##   =   ¡RI and its crosstalk with other membrane proteins. It is noteworthy that a literature spanning 30 years established the Fc¡RI as a model receptor for studying activation-induced changes in receptor diffusion and lipid raft association. Now a combination of high resolution microscopy methods, including immunoelectron microscopy and # {$ ^ #‹$  ! % #  #  spatial and temporal aspects of receptor topography on the mast cell plasma membrane. Physical crosslinking of Fc¡RI with multivalent ligands leads to formation of IgE receptor clusters, termed “signaling patches,” that recruit downstream signaling molecules. However, classes of receptors that engage solely with monovalent ligands can also form distinctive signaling patches. The dynamic relationships between receptor diffusion, aggregation state, clustering, signal initiation and signal strength   $  # "  #  

INTRODUCTION =  # ##   =   ¡RI, is the principal physiological stimulus for mast cell degranulation. This tetrameric receptor is composed of the IgE-binding _ subunit, two a subunits and a tetraspan ` subunit. There are a total of three ITAMs (immunoreceptor tyrosine-based activation motifs), one in the carboxy terminus of the ` subunit and one in each of the paired a subunits. Activation occurs when IgE binds to polyvalent antigens, crosslinking a minimum of two Fc¡RI-IgE complexes thereby initiating a tyrosine kinase cascade that triggers histamine release and de novo synthesis of leukotrienes, prostaglandins1 and an impressive list of cytokines and chemokines.2 Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 91

92

MAST CELL BIOLOGY

Biochemical details of this cascade have been the subject of many reviews, including    ‚! 3 and other chapters in this volume. In the 25 yr period spanning from 1975-2000, the Fc¡RI was the subject of pioneering studies that measured the mobility and behavior of membrane proteins. Emerging biophysical #‹$$#$ ‚!  \##˜‚\™  " after in situ electromigration and time-resolved phosphorescence anisotropy, were used to arrive at estimates of lateral diffusion rates of 1.5-4 = 10<10 cm2/s for resting receptors.4,5 Rotational correlation values ranged from 23-65 ms6-8 with slower values attributable to measurements at low temperatures. In these early studies, incomplete recovery from photobleaching was already recognized as an indication that a small fraction of receptors were immobile in resting cells, with marked increases in the immobile fraction after antibody-mediated crosslinking and the concomitant observation of microaggregates and # !  # {$   4,9 Rotational mobility was also markedly restricted after receptor crosslinking.6 These and related studies led to a number of important hypotheses that are still being vigorously tested today. These include revision of classical Brownian diffusion models of cell membrane proteins to explain restricted lateral mobility10 and the widely-held concept that Fc¡RI immobilization must be necessary for signal initiation.11,12 Antigen valency and structure, particularly as it relates to the orientation and distance of Fc¡RI subunits within aggregates, also continues to be a highly relevant topic13 #    <  #  }    

+*(( +% *^ * & % +%       # {$   #  !  $  % < for plasma membrane structure.14 This model, which integrated many studies from the preceding decade, viewed cellular membranes as two-dimensional solutions composed of integral membrane proteins within a lipid matrix. It proposed that most membrane constituents diffuse rapidly and are randomly arranged, but it acknowledged the possibility of ordered membrane regions based on early EM images of clustered proteins. Variations of this model arose however, beginning with the terms “lipid rafts” and “liquid ordered and disordered domains”.15-17 Seminal studies by several groups working in the mast cell

  !  !  #   ##       $ the recovery of Fc¡RI and its signaling partners or regulators in light fractions of sucrose gradients after stimulation.18-21 While the popularity of the lipid raft concept energized the membrane biology community, artifacts associated with merging of compartments by detergent treatment may have also led to misconceptions. This is now the prevailing view in #   !   !%   # #   #    # likelihood that microdomains are both small, readily exchangeable and complex.22-25 Clearly,   #    $      #      #  An alternative view has re-emerged for membrane organization, referred to as the “protein islands” model.26,27 It is similar to the predictions of Yechiel and Edidin,28 who proposed that “plasma membranes are organized into protein-rich lipid domains, separated by a protein-poor lipid continuum.” Importantly, the protein islands model explains membrane heterogeneity, based upon the strong segregation of distinct protein subpopulations with islands and acknowledges rich association with cholesterol and <  *#  !!    # {$     density on diffusivity,29 #   !#  ^ #  zones transiently trap diffusing membrane proteins on T cells.30

SPA P TIO-TEMPORAL SIGNALING IN MAST CELLS

93

ELECTRON MICROSCOPY PROVIDES A BIRD’S EYE VIEW OF Fc¡RI SIGNALING PATCHES  =    ˜= ™  ! #   $ images of immuno-gold labeled Fc¡RI “patches”,31 that were hinted at in the early ${$  " 4 The size of patches and the time course for their formation, was shown by SEM to be dependent on antigen dose.32 By 2000, our group had developed the technology to prepare native membrane sheets ripped from rat basophilic leukemia (RBL-2H3) cells at various stages of activation.33 With the improved resolution of Transmission Electron Microscopy (TEM), these preparations could be labeled more          # !   $#  ‹$$ dots.34 The sheets also permitted access to the cytoplasmic face of the membrane for labeling intracellular proteins recruited during signaling. Figure 1 provides typical views of Fc¡RI signaling patches before (1A) and after 2 min stimulation with antigens with different valency (1B,C). Cells in Figure 1B were

Figure 1. Electron microscopy reveals the spatial distribution of membrane receptors. A) RBL membrane sheets labeled with immunogold as in reference 33 to show that resting Fc¡RI are found as singlets and in small clusters. Arrow marks a clathrin-coated pit. B) Two min after stimulation with 0.1 +g/ml DNP24^†   $        @™ @ $ %#  \= ^ {"  ! DNP ligand (10 nM) also demonstrate extensive Fc¡RI aggregation. D) Simultaneous activation Fc¡RI (10 nm gold) and FPR (5 nm gold) leads to coclustering of the receptors. Scale bars  100 nm.

94

MAST CELL BIOLOGY

activated with DNP24-BSA (0.1 +g/ml) while cells in Figure 1C were stimulated with a \= ^ {"  ! ~\  ˜•  ™ ~ # !  #   receptors are not completely random (Fig 1A). Small clusters, ranging from 2-5 receptors, are typically found in addition to singlets. Fc¡RI clusters generally increase in size after antigen-mediated crosslinking,32 although the increase is somewhat smaller in response to ligands of low valency or even with low concentrations of polyvalent antigens.35 However, comparison of secretory responses for the two antigen conditions illustrated here (60-70% for DNP24-BSA versus 10-30% for the PEG-trivalent ligand) makes it clear that cluster size does not predict signaling output. To further drive this point home, crosslinking with a panel of divalent antibodies against the Fc¡RI _ subunit yielded similar, small cluster sizes.36 Importantly, only the signaling-competent antibodies showed dissociation from Lyn and recruitment of   $  <   <  #  $ 36 Together with other reports,13 these data support the conclusion that dimerizing agents can activate the Fc¡RI, provided that crosslinking induces appropriate orientation or conformational changes in pairs of receptors. Of note, the large signaling patches induced by multivalent antigen are often bordered by coated pits poised to internalize receptors (Fig. 1B, arrow). This observation is highly reproducible, despite the fact that depletion of clathrin fails to inhibit Fc¡RI endocytosis after activation (ref. 37 and our unpublished results). We speculate that, like the BCR,38 compensatory nonclathrin mediated internalization pathways ensure that highly crosslinked Fc¡RI are removed from the mast cell surface.

GPCRs BOUND TO MONOVALENT LIGANDS ALSO FORM SIGNALING PATCHES The N-formyl peptide receptor (FPR) is naturally co-expressed with the IgE receptor on human basophils. In a study of RBL-2H3 transfectants stably expressing the FPR, this GPCR R was observed to form large clusters in response to addition of monovalent ligand.39 Ligand-bound FPR R cluster rapidly and recruit the heterotrimeric G-protein, Gi. FPR R are slowly endocytosed and, by 5 min, most clusters remaining on the surface contain arrestin and very little Gi. We also found that, when ligands for both FPR R and Fc¡RI were added simultaneously, there was a marked increase in colocalization of the two receptor clusters (Fig. 1E). This study suggested that crosstalk between the tyrosine kinase pathway of the Fc¡RI and the G-protein-coupled pathway of the FPR R occurs locally. Barisas and colleagues have proposed that crosstalk between Fc¡RI and the inhibitory receptor, MAFA, may also occur in rafts or specialized domains.40 Together with evidence for clustering of growth factor receptors.41,42 These data also support the notion that receptor clustering is not solely a property of physical crosslinking by multivalent ligand (as is the case for Fc¡RI) but a more generalized architecture for receptors during active signaling.

SIGNALING PATCHES AS SPECIAL FEATURES OF THE PROTEIN ISLAND NETWORK Experiments comparing the distributions of all proteins in T-cell and RBL membranes shed some light on the issue of colocalizing receptors.26 When bulk

SPA P TIO-TEMPORAL SIGNALING IN MAST CELLS

95

membrane proteins were labeled for electron microscopy with probes targeted at SH-groups, the gold label concentrated in dark regions of membrane that also label for cholesterol. Furthermore, nonraft and raft markers were constructed by expressing tagged versions of N-terminus of Lck with and without mutation of the palmitoylation    $ # ‚§  $   # # %  <  were strongly segregated. These results led us to propose a new model for membrane organization, where proteins organize into complex, cholesterol-rich “islands” in a largely lipid sea. This hypothesis was recently supported by the sophisticated PALM technique (photoactivation localization microscopy).27 We speculate that signaling patches represent specialized domains within islands that initiate, amplify and control signal propagation in a spatially restricted manner.

SINGLE PARTICLE TRAC R KING ALLOWS DIRECT OBSERVATION OF DIFFUSING Fc¡RI We have recently developed a method to directly observe the diffusive behavior of individual Fc¡RI, before and after crosslinking with ligands of different valency. As described in our recent publications,34,35 monovalent Quantum Dot (QD)-labeled IgE can be reliably prepared and used to “prime” Fc¡RI at sparse densities that allow      <  !$ Ÿ^=^  " $ on the surface of live cells. The use of QDs provides several advantages. Their high brightness and photostability allows for long-term single molecule imaging. QDs have broad excitation, yet narrow emission, spectra allowing for simultaneous excitation and      Ÿ $   " %!#   ^ detection. The use of two- or four-color emission beam splitters and sensitive emCCD       %^ !   {$    _^’   distinct probes simultaneously at the single QD level. Using single particle tracking, we have characterized the diffusive behavior of Fc¡‚ ˜ _™ %##    ##       * $       %# «_   $      } $   physiological temperature (35-37°C) as compared to room temperature.34 Even in the resting state, a small fraction of receptors are found to be at least transiently immobile ˜    $   0.001 +m2/s). Individual receptors can switch rapidly and apparently randomly between these diffusion states (see movies in ref 34). The ability to distinguish two or more QD colors was a critical factor in determining whether resting Fc¡RI, observed by EM to be distributed in small clusters, interact %# # # $      ! # $  *#  !  #  %$  #   ‰  Š !       time. As shown in Figure 2C-D, we often observe QD-labeled IgE receptors in close proximity. Figure 2C illustrates a case of two receptors which remain close (500 nm) for several seconds. However, the plot at the right shows that their movements remain uncorrelated. In Figure 2D, two receptors move close to each other for a short time then separate, moving over 2 microns apart before reversing directions and crossing paths again. Analysis of 1,000 close-approach events imaged at 100 frames/sec revealed that correlated motion was not occurring, indicating that the resting receptors do not form a stable complex.34 Thus, the most obvious interpretation is that resting Fc¡‚   { share residency in the same microdomain, but that their associations (if any) during this

96

MAST CELL BIOLOGY

Figure 2. Characterizing Fc ¡RI diffusion. A) Example trajectories of individual Fc¡RI motion as determined by single quantum dot tracking. The expected four modes of motion are observed: free, restricted, directed and immobile (see Andrews et al, 2008). B) Distribution of the modes of motion under different conditions. C,D) Two-color imaging allows us to simultaneously track receptors labeled with QD655-IgE and QD585-IgE. Note that receptors often maintain close proximity but do not demonstrate     # #   "  $         receptor interactions. (C,D from reference 34) E) Example simulation from the Constrained Brownian Motion algorithm. Proteins in the simulation are considered as individual particles or agents that move at each time step (see ref. 43). The membrane can be further subdivided into domains that are lipid-rich (fast diffusion for proteins, white space) and protein-rich (2-fold slower diffusion for proteins in the simulation, grey regions). Slower movement of receptors through the protein-rich domains leads to clustering of resting receptors (F) similar to that seen in electron microscopy images (Fig. 1).

SPA P TIO-TEMPORAL SIGNALING IN MAST CELLS

97

residency period are very weak. This is consistent with time-resolved phosphorescence polarization studies, which concluded that the rotational mobility of resting receptors is neither hindered by direct association with the cytoskeleton or by aggregation.6

MODELING THE INS AND OUTS OF RESTING RECEPTOR R CLUSTERS We have used stochastic modeling approaches to test the possibility that transient residency of resting receptors in islands or rafts provides a plausible explanation for their nonrandom distribution. For these simulations, the 2-D membrane is represented by a Cartesian plane with a periodic boundary such that receptors that leave the plane re-enter on the opposite side of the membrane. Species in the simulation are considered as individual particles or agents that move at each time step under a Constrained Brownian Motion algorithm.43 The membrane can be further subdivided into domains that are lipid-rich (fast diffusion for proteins) and protein-rich (2-fold slower diffusion for proteins  # $™ ˜ _=™ #$# %   # #‹$  # = ‚ # approach is applicable to virtually any resting membrane receptor (including Fc¡RI)  ! $    "    =!       # simulation at random locations, a pattern of clustering will form that closely matches that observed by immunoelectron microscopy and similarly passes the Hopkins test (Fig. 2F). We conclude that, even when receptors are in constant motion, short duration trapping in protein-rich microdomains (islands) can result in clustering. Consistent with this hypothesis is prior evidence that Fc¡RI diffusion is profoundly {$     % *#  29 found that Fc¡‚ %   %#  $  # !  ‚†ƒ  "      # $    %  !       #< RBL cells, which released constraints of the cortical cytoskeleton. Other experiments during the early 1990s demonstrated that crowding slows diffusion of membrane receptors      44,45 ˆ $    <  "     {$  receptor diffusion, by serving as physical barriers or corrals34 and by anchoring protein islands.26 The cytoskeleton will be further discussed in sections below. IMMOBILIZATION OF CROSSLINKED FC¡RI IS DEPENDENT ON DOSE AND ANTIGEN VALENCY * <  Ÿ^= $     $  #     ! the transition from resting to crosslinked receptors in real time (Fig. 3A). As predicted, based upon prior reports of Fc¡RI immobilization after crosslinking,4,11,46 we observed that stimulation with modest to high doses of highly polyvalent antigen (DNP24-BSA, 0.1-10 +g/ml) resulted in rapid immobilization (Fig. 3A). Our real-time assay was directly able to determine the kinetics of this change in mobility, found to occur in less than 20 s. Somewhat unexpectedly, we found that low doses of the same highly multivalent antigen failed to induce immobilization despite robust stimulation of degranulation.35 We similarly found that even high doses of low valency antigens (DNP2-BSA, DNP4-BSA), also shown to induce degranulation, did not cause receptor immobilization (Fig. 3B). *# $    # $ # }    ‹$ ! for signal initiation from the crosslinked Fc¡RI. However, it is intriguing to consider this

98

MAST CELL BIOLOGY

Figure 3. A) Plot of the average displacement of QD-IgE-Fc¡RI complexes between subsequent frames over the time course of a single movie. After addition of 1 +g/ml DNP24-BSA, the displacement is seen to rapidly decrease, indicating a slow-down in receptor mobility (see ref. 34). Red line is 10   !     "   #  †™ @$$! \      # $   ˜™  Ÿ^=^¡RI before and after crosslinking with antigens of different valency. Note that with DNP2-BSA (red) receptor mobility did not change from resting (black) even though degranulation was seen under these conditions. Higher valencies did cause receptor slow-down, presumably due to the formation of larger aggregates. (Reproduced with premission from: Andrews NL et al. Immunity 2009; 31(3):469-479;35 ©2009 Elsevier.) C) Tracking of QD-IgE-Fc¡RI   "  \^ ˜ ™ #% # #   ‘  ˜  ™     #  < ˜ ™ `   %    %# \  # } #   ˜ ref. 34). Scale bar, 1um. D) AFM image revealing the topography of the cytoplasmic face of an RBL membrane sheet. The image is pseudo-colored to indicate heights ranging from 5-6 nm (dark red, the width of a lipid bilayer) to 30 nm (hot yellow) above the substrate. Raised domains dot the landscape       #  ˜‚ $ %#      < >   †# J 2006; 90(7):2404-2413;58 ©2006 Elsevier.)

 #   "   !$ % < $  $$       } antibodies for Fc¡RI stimulation (reviewed in see ref. 13). There are strong correlations between signaling potency and the predicted oligomeric state of crosslinked receptors. Dimerizing reagents, including both divalent ligands and antibodies directed at either the Fc¡RI _ subunit or at IgE, exhibit a wide range of stimulatory capacity. It is possible for such reagents to induce immobilization in the absence of signaling, at least as measured by

SPA P TIO-TEMPORAL SIGNALING IN MAST CELLS

99

degranulation and Ca2 mobilization.47 †!  %# {" \= ^   fail to stimulate measurable secretion48 and can even be inhibitory.49 In general, higher oligomers induced by higher valency antigens—or further crosslinking of antibody-bound complexes—generate better signaling from the RBL-2H3 cell line over a broad dose response range. A number of factors have been implicated, including overriding constraints imposed by cyclic dimers as well as the improved geometry, spacing and orientation of receptors in large aggregates (see discussion in ref. 13). Lateral forces may also be a factor, based upon observations that liposomes or lipid bilayers bearing haptens for IgE can recruit large numbers of receptors to the adhesive surface and can induce weak but measurable secretory responses50,51 IgE-Fc¡RI complexes engaged by lipid-conjugated monovalent hapten form a “synapse” and induce degranulation while remaining highly mobile, further demonstrating that immobilization is not required for signal initiation.52 We note that maximal secretory responses of bone marrow derived mast cells (BMMC) and human basophils occur over a much narrower range even for the highly polyvalent antigens,35,53 often abruptly dropping off at modest concentrations of crosslinking reagents. This phenomenon has been referred to as “high dose inhibition” and may be attributed to active negative regulatory mechanisms that dominate in Fc¡RI-bearing primary cells. In support of this, we found that receptor immobilization can trigger internalization even in the absence of signaling.35

THE INFLUENCE OF THE CORTICAL CYTOSKELETON The actin cytoskeleton has long been proposed to form “corrals” or “picket fences” capable of restricting movements of membrane proteins.54-56 By coupling QD-IgE single    < %# *‚ ˜   {  ™ % %    directly test this widely-held theory.34 To perform this series of experiments, RBL cells were selected that stably express GFP-actin. The movements of resting QD-labeled IgE   %  #  < !     # # #  $   live cells settled on coverslips. Images were captured at a rate of 35-100 frames/second. As illustrated in Figure 3C, QD-labeled receptors were shown to be markedly restricted by the network of actin cables juxtaposed to the inner membrane. Moreover, because the actin network is dynamic, the shapes of the corrals were shown to be constantly changing and therefore frequently permit QD-labeled receptors to “slip” between openings to previously inaccessible compartments. The GFP-actin cables, seen by TIRF, span distances of several microns. We have $ ## $   #‹$  $       <  #      !  # {$    !  *‚  that helps to eliminate out-off focus light.57,58 The image in Figure 3D shows the topographic detail of the cytoplasmic face of a hydrated membrane sheet prepared from an RBL cell. It was acquired on the AFM (Atomic Force Microscope) of our collaborator, Alan Burns, at Sandia National Laboratory. The image is pseudo-colored to indicate heights ranging from 5-6 nm (dark red, the width of a lipid bilayer) to 30 nm (hot yellow) above the $  ‚   #        #  This image and others obtained on the transmission electron microscope (see cables in the images in Fig. 1), led us to conclude that the cortical cytoskeleton links protein islands in the RBL membrane. We found that labels for actin and myosin do not uniformly label #  #% <57 Instead actin and myosin are usually concentrated at junctions in

100

MAST CELL BIOLOGY

the network. This leads us to speculate that actin bundles are only a minor fraction of the network, providing an explanation for the fact that latrunculin treatment only modestly enhances the mobility of molecules in single particle tracking experiments.59,34 Lillemeier and colleagues26 found that protein islands are smaller but persistent in T-cell membranes when their cytoskeletal tethers are disrupted by latrunculin treatment. Dense cytoskeletal networks or direct associations with crosslinked Fc¡RI may actually limit the signaling process, as suggested by the work of Seagrave and Oliver32 and Holowka and Baird60 Seagrave and Oliver61 noted that cytochalasin treatment reduced the size of Fc¡RI aggregates observed by SEM, leading to the hypothesis that very large aggregates signal poorly.

INTRAC R ELLULAR PROTEIN-PROTEIN INTERAC R TIONS PROVIDE AN ADDITIONAL MECHANISM TO STABILIZE SIGNALING PATCHES: THE CASE OF LAT Like other immunoreceptors, signal propagation following Fc¡RI crosslinking involves the recruitment of a large number of proteins that contain binding motifs, such as SH2 (src homology 2), SH3 (src homology 3), PH domains, PTB domains and others.3,62 Large macromolecular complexes can form based on these interactions, with the potential to induce aggregation and stabilize clusters of receptors and other signaling proteins. Goldstein and colleagues have used mathematical modeling to validate intracellular cross-bridging as a mechanism for growth and stabilization of large clusters of the scaffolding protein, Linker for Activation of T cells (LAT).63 The model is based upon the ability of Grb2 to bind via its SH2 domain to phosphorylated LAT and to two molecules of SOS through its two SH3 domains.64,65 Thus multivalent, cooperative protein-protein interactions that propagate at the cytoplasm-membrane interface may explain, in large part, the observation that LAT clusters markedly increase in size following Fc¡RI crosslinking57 or TCR R activation.27,64 It seems likely that this paradigm will be broadly applicable to other transmembrane proteins that participate in complex signaling cascades.

WHAT ABOUT LIPIDS? The local lipid environment of immunoreceptors remains enigmatic. In 2002, Jacobson and Anderson proposed that membrane proteins might be surrounded by a lipid shell.66 Proof for this attractive concept has eluded investigators to date, mostly because of technical limitations. However, evidence for strong lipid association with Fc¡RI dates back to the work of Rivnay, Metzger and colleagues.67,68 The addition of lipids to detergent micelles delayed the slow dissociation of Fc¡RI subunits, with the best protection provided by dipalmitoylglycerol and cholesterol.69 If a lipid shell envelopes Fc¡RI, cholesterol is perhaps our best candidate. However, results from multiple laboratories remain complex and, in our opinion, controversial. For example, RBL cells depleted of cholesterol after mBCD treatment show profound      # $ %  ‰{Š #     $ overall height of all membrane domains.58 *#    <   $     differing reports that cholesterol depletion either inhibits the entire Fc¡RI tyrosine kinase cascade70 or has little effect on Fc¡RI-induced phosphorylation but differential effects on calcium mobilization and degranulation.71,72

SPA P TIO-TEMPORAL SIGNALING IN MAST CELLS

101

Figure 4. A) Pseudo-colored X-ray spectral electron microscopy image, where arrows point to “hot spots” for osmium staining over a region of a membrane sheet. Images were captured on a Phillips FEI Tecnai F30-ST equipped with an EDAX r-TEM SUTW energy dispersive X-ray detector. (Reproduced with permission from: Pike LJ et al. J Biol Chem 2005; 280(29):26796-26804.75). B) Results from lipid analysis of IgE-containing vesicles (red) compared to Thy-1-containing vesicles (blue). Lipid content in IgE-vesicles was 50% cholesterol, compared to less than 25% in Thy-1 vesicles. C) Simultaneous imaging of cholesterol-18 FITC (green) and IgE-Fc¡RI (red) in the membrane of an RBL cells as it contacts a lipid bilayer containing monovalent DNP. Cholesterol and Fc¡RI are seen to colocalize in patches that travel together during synapse formation. 52

We have focused on alternative approaches to evaluate the lipid environment of Fc¡RI in resting and activated states. We previously made use of the capabilities of X-ray spectral electron microsopy,73 %#      #   #   X-rays when excited by the electron beam and the intensity of the signal relates to its concentration. Data can be acquired in scanning mode on STEM instruments, reporting "^^" !$       $–    *=

mode, one also can acquire a more traditional image of the sample that is based on contrast. We focused on the distribution of osmium, a contrast agent commonly used in electron microscopy due to its ability to react with double bonds in lipids. As shown in Figure 4A, this method revealed that membranes concentrate osmium in patches on the membrane; and this observation strongly suggested that lipids with double bonds (a category that includes cholesterol) were unequally distributed.

102

MAST CELL BIOLOGY

Our next approach was to isolate plasma membrane-derived vesicles for lipidomics analysis in collaboration with Robert Murphy (Univ. of Colorado), with a primary goal of avoiding detergent expected to disrupt native structure. Our strategy was to crosslink =    \^#  *#^  # $   ‚†ƒ  %#    forming large patches enriched in either activated receptors or aggregated Thy-1.74 Cells were rapidly chilled and mechanically disrupted to vesiculate the plasma membrane. After a series of steps to reduce contamination from unbroken cells, etc, we then used immunomagnetic bead isolation to isolate right-side-out vesicles containing IgE receptors  *#^ = # %    _^   #  ˜ !$ #   content) and then samples were prepared and sent to Denver. This process was repeated over several years for an in depth analysis of the levels of fatty acid saturation, cholesterol !    *# $   !     ˜™ †# vesicle preparations contained a complex set of plasma membrane phospholipids, as analyzed by mass spec analysis; (2) Lipid content in vesicles immuno-isolated with anti-IgE was 50% cholesterol, compared to less than 25% in Thy-1 vesicles (Fig. 4B); (3) Less than 50% of the fatty acids in either domain were saturated, inconsistent with the concept of liquid ordered domains; and (4) IgE receptor-containing vesicles contained 2-3 times   #   # # *#^  *#   is particularly intriguing, since the vinyl ether linkage at the sn-1 position introduces a double bond that is very near the lipid head group. Plasmalogens were also found to be enriched in lipid raft fractions containing the EGFR.75 Although plasmalogens are %  $  $    #      76 Remarkably, % $  %# #    #! !   $            @~  *#   have been linked to impaired intracellular cholesterol distribution, plasma membrane function and ER/Golgi R structural changes (reviewed in see ref. 76). On an interesting      # ^  ƒˆ  "# #  $  constitutive cytokine production, which may be linked to down regulation of Lyn acting in its negative regulatory role.77 Recently we employed new strategies to allow us to track cholesterol in live cells. & <%#>  *   ~ ƒ  % #!! {$  cholesterol derivatives in RBL-2H3 cells. Our best success is with cholesterol-18 FITC that has a long linker for the chromophore (Fig. 4C). This probe is easily tracked when imaging in TIRF mode and forms patches that markedly travel and colocalize with Fc¡RI recruited to lipid bilayers presenting monovalent, mobile ligands.53 These patches are remarkably similar to the microclusters of TCR R and BCR R that travel to the well described synapses of T cells78 and B cells.79 *# % #  ! #  $   immunoreceptors are enriched in cholesterol as they move to the synapse. It remains to be determined which of cholesterol’s biochemical and biophysical properties predominate in the regulation of membrane architecture: its ability to {$     {$–     #  $  phosphatidylserine;80           –81,82 or its importance for membrane curvature.83 For the Fc¡RI, we are struck by the enigma of its association with mobile, but not immobile, receptors at the immune synapse.52 If cholesterol comprises part of the lipid shell around Fc¡‚  $  $  %< #  $  if receptors become trapped by binding to an immobile ligand. Since receptors trapped in this way cause massive degranulation, it also appears that cholesterol is not an absolute requirement for signal transduction to commence.

SPA P TIO-TEMPORAL SIGNALING IN MAST CELLS

103

CONCLUSION AND FUTURE DIRECTIONS As the entry point for signaling input, the plasma membrane continues to be the focus of intense study. Its constituents occupy a surprisingly complex 2-D environment, %#        $         spaces. Perhaps the greatest next challenge is to capture the subsecond temporal details of signal transducers recruited to Fc¡‚   $ %#     well as to downstream signaling scaffolds such as LAT. This will be an important goal, since population readouts such as phosphorylation patterns of Fc¡RI`, Syk and LAT fail to correlate well with downstream responses such as degranulation.84 We anticipate the development of such measurements in the near future, since innovation has been a hallmark of the mast cell imaging community, often leading # %  #       =" $ # !  GFP/SH2 and GFP-PH domain fusion proteins as reporters of protein translocation to the plasma membrane probes in response to Fc¡RI activation.85 Fluorescence correlation spectroscopy (FCS) has been creatively applied by the collaborative team of Webb, Baird and Holowka86 to measure association between Fc¡RI and Lyn tyrosine kinase on live cells following antigen stimulation. This important subject deserves revisiting, since the authors’ choice of room temperature measurements (to slow  }    ${™ <   #    $   Lyn recruitment (5-6 min after antigen addition). Our group has developed multi-color quantum dot probes to directly observe the formation of Fc¡RI aggregates, to track the correlated motion of small aggregates and to observe the abrupt immobilization of larger aggregates.34,35 FCS and SPT represent powerful technologies for measuring the dynamics of protein-protein interactions. In addition, emerging super-resolution and correlation imaging technologies hold much promise for the quantitative measurement of protein-protein interactions in living cells (reviewed in see ref. 87). Such advances in imaging techniques will allow us to take the next step—beyond the plasma membrane—to look at the cytoplasmic protein dynamics and interactions that propagate the message initiated by Fc¡RI aggregation.

ACKNOWLEDGEMENTS This work was supported by NIH R01AI051575 (BSW), NIH RO1GM49814 (JMO), the Human Frontiers Science Program (DSL), NIH SPORE M01RR000997 and NIH P50GM085273 (that supports the New Mexico Spatiotemporal Modeling Center). We acknowledge important institutional resources, particularly the UNM Cancer Center Fluorescence Microscopy Facility and the Electron Microscopy Facility. We acknowledge present and former members of the Cell Pathology laboratory for their important contributions, particularly Mary Ann Raymond-Stintz, Angela Welford, Nicholas Andrews, Janet Pfeiffer, Zurab Surviladze, Genie Hsieh, Mei Xue and Amanda Carroll-Portillo whose work is included in this summary. We also thank our collaborators at UNM (Keith Lidke, Stanly Steinberg, Jeremy Edwards), Sandia National Laboratory (Alan Burns, Jerilyn Timlin, Paul Kotula), Los Alamos National Laboratory (Byron Goldstein, Bill Hlavacek), University of Notre Dame (Basar † ™  '!   @  ˜‚  $ #™     $ and valuable discussion.

104

MAST CELL BIOLOGY

REFERENCES 1. Wasserman SI. Basic mechanisms in asthma. Ann Allergy 1988; 60(5):477-482. _   ƒ *  @ #$       {      #  #    $‘    ##^  =      #   Immunol 2008; 145(3):182-192. Œ     ‚!  > *#   < % < $   ! $ ‚! _••€– 228(1):149-169. 4. Schlessinger J, Metzger H, Webb WW et al. Lateral motion and valence of Fc receptors on rat peritoneal mast-cells. Biophys J 1977; 17(2):A72-A72. 5. Mccloskey MA, Liu ZY, Poo MM. Lateral electromigration and diffusion of Fc-epsilon receptors on rat basophilic leukemia-cells—effects of IgE binding. J Cell Biol 1984; 99(3):778-787. 6. Zidovetzki R, Bartholdi M, Arndtjovin D et al. Rotational-dynamics of the Fc receptor for immunoglobulin-e on histamine-releasing rat basophilic leukemia-cells. Biochemistry 1986; 25(15):4397-4401. ” \#  ˆ  = >! *  ‚^  # ^   ^      monoclonal-antibodies and IgE. Biochemistry 1991; 30(14):3450-3458. 8. Myers JN, J Holowka D, Baird B. Rotational motion of monomeric and dimeric immunoglobulin E-receptor complexes. Biochemistry 1992; 31(2):567-575. 9. Ryan R TA, Myers J, Holowka D et al. Molecular crowding on the cell surface. Science 1988; 239(4835):61-64. 10. Feder TJ, BrustMascher I, Slattery JP et al. Constrained diffusion or immobile fraction on cell surfaces: A new interpretation. Biophys J 1996; 70(6):2767-2773. 11. Menon AK, Holowka D, Webb WW et al. Cross-linking of receptor-bound IgE to aggregates larger than dimers leads to rapid immobilization. J Cell Biol 1986; 102(2):541-550. 12. Tamir I, SchweitzerStenner R, Pecht I. Immobilization of the type I receptor for IgE initiates signal transduction in mast cells. Biochemistry 1996; 35(21):6872-6883. Œ `%<    *  @   #  $$ =      $$    ligands. Immunol Rev 2007; 217:269-279. ’  > ~ ƒ *# {$    #  $$       €”_– 175(23):720-731. 15. Brown DA, Rose JK. Sorting of gpi-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell-surface. Cell 1992; 68(3):533-544. 16. Simons K, Ikonen E. Functional rafts in cell membranes. Nature 1997; 387(6633):569-572. 17. Ahmed SN, Brown DA, London E. On the origin of sphingolipid/cholesterol-rich detergent-insoluble cell membranes: Physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 1997; 36(36):10944-10953. 18. Field KA, Holowka D, Baird B. Fc-epsilon-ri-mediated recruitment of P53/56 (Lyn) to detergent-resistant membrane domains accompanies cellular signaling. Proc Natl Acad Sci USA 1995; 92(20):9201-9205. €   `%<  †  † @ } !  # ##   $$ =  within membrane domains. J Biol Chem 1997; 272(7):4276-4280. 20. Field KA, Holowka D, Baird B. Structural aspects of the association of Fc epsilon RI with detergent-resistant membranes. J Biol Chem 1999; 274(3):1753-1758. 21. Draberova L, Draber P. Thy-1 glycoprotein and src-like protein-tyrosine kinase P53/P56 (Lyn) are associated in large detergent-resistant complexes in rat basophilic leukemia-cells. Proc Natl Acad Sci USA 1993; 90(8):3611-3615. 22. Vereb G, Szollosi J, Matko J et al. Dynamic, yet structured: the cell membrane three decades after the singer-nicolson model. Proc Natl Acad Sci USA 2003; 100(14):8053-8058. 23. Lagerholm BC, Weinreb GE, Jacobson K et al. Detecting microdomains in intact cell membranes. Annu Rev Phys Chem 2005; 56:309-336. 24. Hancock JF. Lipid rafts: contentious only from simplistic standpoints. Nat Rev Mol Cell Biol 2006; 7(6):456-462. 25. Jacobson K, Mouritsen OG, Anderson RG. Lipid rafts: at a crossroad between cell biology and physics. Nat Cell Biol 2007; 9(1):7-14. 26. Lillemeier BF, Pfeiffer JR, Surviladze Z et al. Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proc Natl Acad Sci USA 2006; 103(50):18992-18997. 27. Lillemeier BF, Mortelmaier MA, Forstner MB et al. TCR R and Lat are expressed on separate protein islands on T-cell membranes and concatenate during activation (vol 11, pg 90, 2010). Nat Immunol 2010; 11(6):543-543. _— š# = =    ^     ^  > @ † €—”– 105(2):755-760. 29. Thomas JL, Feder TJ, Webb WW. Effects of protein-concentration on ige receptor mobility in rat basophilic leukemia-cell plasma-membranes. Biophys J 1992; 61(5):1402-1412. 30. Douglass AD, Vale R RD. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T-cells. Cell 2005; 121(6):937-950.

SPA P TIO-TEMPORAL SIGNALING IN MAST CELLS

105

31. Stump RF, Pfeiffer JR, Seagrave J et al. Mapping gold-labeled ige receptors on mast-cells by scanning electron-microscopy—receptor distributions revealed by silver enhancement, backscattered electron imaging and digital image-analysis. J Histochem Cytochem 1988; 36(5):493-502. 32. Seagrave J, Pfeiffer JR, Wofsy C et al. Relationship of ige receptor topography to secretion in Rbl R -2h3 mast-cells. J Cell Physiol 1991; 148(1):139-151. 33. Wilson BS, Pfeiffer JR, Oliver JM. Observing FcepsilonRI signaling from the inside of the mast cell membrane. J Cell Biol 2000; 149(5):1131-1142. 34. Andrews NL, Lidke KA, Pfeiffer JR R et al. Actin restricts Fc epsilon RI diffusion and facilitates antigen-induced receptor immobilization. Nat Cell Biol 2008; 10(8):955-963. 35. Andrews NL, Pfeiffer JR, Martinez AM et al. Small, mobile Fc epsilon R1 receptor aggregates are signaling competent. Immunity 2009; 31(3):469-479. 36. Lara M, Ortega E, Pecht I et al. Overcoming the signaling defect of Lyn-sequestering, signal-curtailing FcepsilonRI dimers: aggregated dimers can dissociate from Lyn and form signaling complexes with Syk. J Immunol 2001; 167(8):4329-4337. Œ” <#!    †     *# ##^  $$^=  ˜  ‚™     \^_# ^ ^ # *   _••“– ”˜“™“”Œ^“— 38. Stoddart A, Jackson AP, Brodsky FM. Plasticity of B-cell receptor internalization upon conditional depletion of clathrin. Mol Biol Cell 2005; 16(5):2339-2348. Œ€ $  `#  ‚^}    ! ~^     ##^  = receptor occupy common domains for signaling and internalization. Mol Biol Cell 2007; 18(4):1410-1420. 40. Barisas BG, Smith SM, Liu JJJ et al. Compartmentalization of the Type I Fc epsilon receptor and MAFA on mast cell membranes. Biophys Chem 2007; 126(1-3):209-217. 41. Nagy P, Jenei A, Kirsch AK K et al. Activation-dependent clustering of the erbB2 receptor tyrosine kinase     ^     > @  €€€– _ ˜ \ ™”ŒŒ^”’ 42. Yang S, Raymond-Stintz MA, Ying W et al. Mapping ErbB receptors on breast cancer cell membranes during signal transduction. J Cell Sci 2007; 120(Pt 16):2763-2773. 43. Hsieh G, Yang S, Raymond-Stintz M et al. Stochastic simulations of ErbB homo and hetero-dimerization: potential impacts of receptor conformational Ctate and spatial segregation. IET Systems Biology 2008; Invited submission(q-Bio Special issue). 44. Tank DW, Wu ES, Webb WW. Enhanced molecular diffusibility in muscle membrane blebs—release of lateral constraints. J Cell Biol 1982; 92(1):207-212. 45. Peters R, Cherry RJ. Lateral and rotational diffusion of bacteriorhodopsin in lipid bilayers—experimental test of the saffman-delbruck equations. Proc Natl Acad Sci USA-Biological Sciences 1982; 79(14):4317-4321. 46. Menon AK, Holowka D, Webb WW et al. Clustering, mobility and triggering activity of small ligomers of immunoglobulin-E on rat basophilic leukemia-cells. J Cell Biol 1986; 102(2):534-540. 47. Posner RG, Subramanian K, Goldstein B et al. Simultaneous cross-linking by 2 nontriggering bivalent ligands causes synergistic signaling of ige Fc-epsilon-Ri complexes. J Immunol 1995; 155(7):3601-3609. 48. Posner RG, Geng D, Haymore S et al. Trivalent antigens for degranulation of mast cells. Org Lett 2007; 9(18):3551-3554. ’€ †  => `%<  @ &   `# ! ˜# ™  #$     inhibition of immune receptor activation. Biochemistry 2003; 42(44):12739-12748. 50. Mcconnell HM, Watts TH, Weis R RM et al. Supported planar membranes in studies of cell-cell recognition in the immune-system. Biochim Biophys Acta 1986; 864(1):95-106.  <  \ @^$  $  ^¢$$ and development of adhesion. J Cell Biol 1986; 102(6):2185-2196. 52. Carroll-Portillo A, Spendier K, Pfeiffer J et al. Formation of a mast cell synapse: Fc epsilon RI membrane dynamics upon binding mobile or immobilized ligands on surfaces. J Immunol 2010; 184(3):1328-1338. Œ  @ƒ & † ˆ! >     #   ‚^!   <  <  zap-70 in human basophils. J Allergy Clin Immunol 1998; 102(2):304-315. 54. Sheetz MP. Membrane skeletal dynamics—role in modulation of red-cell deformability, mobility of transmembrane proteins and shape. Semin Hematol 1983; 20(3):175-188. 55. Saxton MJ. Single-particle tracking—effects of corrals. Biophys J 1995; 69(2):389-398. 56. Kusumi K A, Ike H, Nakada C et al. Single-molecule tracking of membrane molecules: plasma membrane compartmentalization and dynamic assembly of raft-philic signaling molecules. Semin Immunol 2005; 17(1):3-21. 57. Wilson BS, Pfeiffer JR, Surviladze Z et al. High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT. J Cell Biol 2001; 154(3):645-658. 58. Frankel DJ, Pfeiffer JR, Surviladze Z et al. Revealing the topography of cellular membrane domains by      {$   †# > _••“– €•˜”™_’•’^_’Œ 59. Fujiwara T, Ritchie K, Murakoshi H et al. Phospholipids undergo hop diffusion in compartmentalized cell membrane. J Cell Biol 2002; 157(6):1071-1081.

106

MAST CELL BIOLOGY

“• \  ƒ `  ~* `%<    =! $         $ # coupling between poorly dissociable IgE-Fc epsilon RI aggregates and downstream signaling pathways. Biochemistry 1997; 36(24):7447-7456. 61. Seagrave J, Oliver JM. Antigen-dependent transition of ige to a detergent-insoluble form is associated with reduced ige receptor-dependent secretion from Rbl R -2h3 mast-cells. J Cell Physiol 1990; 144(1):128-136. 62. Zhang J, Leiderman K, Pfeiffer JR R et al. Characterizing the topography of membrane receptors and signaling molecules from spatial patterns obtained using nanometer-scale electron-dense probes and electron microscopy. Micron 2006; 37(1):14-34. 63. Nag A, Monine MI, Faeder JR R et al. Aggregation of membrane proteins by cytosolic cross-linkers: theory and simulation of the LAT-Grb2-SOS1 system. Biophys J 2009; 96(7):2604-2623. 64. Bunnell SC, Singer AL, Hong DI et al. Persistence of cooperatively stabilized signaling clusters drives T-cell activation. Mol Cell Biol 2006; 26(19):7155-7166. 65. Houtman JC, Yamaguchi H, Barda-Saad M et al. Oligomerization of signaling complexes by the multipoint R to both LAT and SOS1. Nat Struct Mol Biol 2006; 13(9):798-805. binding of GRB2 66. Anderson RG, Jacobson K. A role for lipid shells in targeting proteins to caveolae, rafts and other lipid domains. Science 2002; 296(5574):1821-1825. 67. Rivnay B, Wank SA, Poy G et al. Phospholipids stabilize the interaction between the alpha-subunit and beta-subunit of the solubilized receptor for immunoglobulin-E. Biochemistry 1982; 21(26):6922-6927. 68. Rivnay B, Fischer G. Phospholipid distribution in the microenvironment of the immunoglobulin E-receptor from rat basophilic leukemia-cell membrane. Biochemistry 1986; 25(19):5686-5693. 69. Kinet JP, Quarto R, Perezmontfort R et al. Noncovalently and covalently bound lipid on the receptor for immunoglobulin-E. Biochemistry 1985; 24(25):7342-7348. 70. Sheets ED, Holowka D, Baird B. Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation of Fc epsilon RI and their association with detergent-resistant membranes. J Cell Biol 1999; 145(4):877-887. 71. Surviladze Z, Harrison KA, Murphy RC et al. Fc epsilon RI and Thy-1 domains have unique protein and lipid compositions. J Lipid Res 2007; 48(6):1325-1335. 72. Yamashita T, Yamaguchi T, Murakami K et al. Detergent-resistant membrane domains are required for mast  ! $     ##  $    # ##    for IgE. J Biochem 2001; 129(6):861-868. 73. Wilson BS, Steinberg SL, Liederman K et al. Markers for detergent-resistant lipid rafts occupy distinct and dynamic domains in native membranes. Mol Biol Cell 2004; 15(6):2580-2592. 74. Surviladze Z, Harrison KA, Murphy RC et al. FcepsilonRI and Thy-1 domains have unique protein and lipid compositions. J Lipid Res 2007; 48(6):1325-1335. 75. Pike LJ, Han X, Gross R RW. Epidermal growth factor receptors are localized to lipid rafts that contain a balance    $ {   #$  $ > † @# _••– _—•˜_€™_“”€“^_“—•’ ”“    *  ‘!    *# # ^  $  < %  functions. Biochim Biophys Acta 2006; 1763(12):1511-1526. ”” ! !  & @ ˆ    @#      $   #^^}   reveals increased mast cell responsiveness. J Exp Med 2006; 203(5):1161-1171. 78. Dustin ML. A dynamic view of the immunological synapse. Semin Immunol 2005; 17(6):400-410. 79. Tolar P, Hanna J, K Krueger PD et al. The constant region of the membrane immunoglobulin mediates B-Cell-Receptor clustering and signaling in response to membrane antigens. Immunity 2009; 30(1):44-55. 80. Sprong H, van der Sluijs P, van Meer G. How proteins move lipids and lipids move proteins. Nat Rev Mol Cell Biol 2001; 2(7):504-513. 81. Thiele C, Hannah MJ, Fahrenholz F et al. Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles. Nat Cell Biol 2000; 2(1):42-49. 82. Epand RM. R Cholesterol and the interaction of proteins with membrane domains. Prog Lipid Res 2006; 45(4):279-294. 83. Huttner WB, Zimmerberg J. Implications of lipid microdomains for membrane curvature, budding and

¢  @$ ˆ @ † _••– Œ˜’™’”—^’—’ 84. Paar JM, Harris NT, Holowka D et al. Bivalent ligands with rigid double-stranded DNA spacers reveal structural constraints on signaling by Fc epsilon RI. J Immunol 2002; 169(2):856-864. 85. Stauffer TP, Meyer T. Compartmentalized IgE receptor-mediated signal transduction in living cells. J Cell Biol 1997; 139(6):1447-1454. 86. Larson DR, Gosse JA, Holowka DA et al. Temporally resolved interactions between IgE receptors and Lyn <  !  $   _^# {$          2007; 13:8-9. 87. Lidke DS, Wilson BS. Caught in the act: quantifying protein behaviour in living cells. Trends Cell Biol 2009; 19(11):566-574.

CHAPTER R7 THE MECHANISMS OF EXOCYTOSIS IN MAST CELLS Ulrich Blank Inserm U699, Paris, France; and Faculté de Médecine Paris Diderot–Site Xavier Bichat, Université Paris 7, Paris, France  8& " <

Abstract:

' ! # $# ##   =   ˜¡RI), mast cells (MCs) can release up to 100% of their content of preformed mediators stored in cytoplasmic secretory granules by compound exocytosis. This causes Type I immediate hypersensitivity reactions and, in the case of inappropriate activation by allergens, the symptoms of allergy. Recent work has uncovered a central role of SNARE (Soluble NN ethylmaleimide-Sensitive Factor (NSF) Attachment Protein (SNAP) Receptors) proteins in regulating the numerous membrane fusion events during " *# #       % $    @ " # participate in the regulation of membrane fusion and the connection of the fusion machinery with early signaling events. The purpose of this chapter is to describe these proteins and provide a brief overview on their mechanism of action.

INTRODUCTION

@   }     #   {   %# # !     %   {   $  < # proteases and proteoglycans, are stored in cytoplasmic secretory granules (SGs) and can be released by exocytosis upon stimulation, ensuring an immediate and maximal biologic effect. They also newly synthesize arachidonic acid metabolites (LTC4, PGD2), as well  ! $ {  <#<   !$   1 Together these account for the biological activities of MCs in innate and adaptive immunity. While physiologic functions of MCs have been increasingly recognized, they are notoriously <%  #      # {   *#  

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 107

108

MAST CELL BIOLOGY

understanding the mechanisms of mediator secretion is of prime interest for therapeutic     ! #!     #     #    # ##     = ˜¡RI).1 This includes knowledge $ #  ! !!  #    $  $  "  # become clear that they rely on the evolutionary conserved machinery of SNARE fusion proteins as well as associated accessory proteins. This chapter describes and discusses the function of this molecular machinery.

REGULATED EXOCYTOSIS IN MAST CELLS While all eukaryotic cells are capable of transporting and exocytosing proteins along the constitutive secretory pathway, some specialized secretory cells including MCs are capable of regulated exocytosis. They store a wide range of secretory products in cytoplasmic SGs, making them available for immediate release upon stimulation. Generally, exocytosis observed in neuronal, neuroendocrine or exocrine cells, involves the fusion of an individual SG with the plasma membrane (PM). For example, at the neuronal synapse this system is highly specialized and coupled to rapid endocytosis and regeneration of secretion-competent SGs within a second, which enables repetitive exocytosis.2 By contrast, MCs are specialized to release virtually all granular content in a single stimulatory event, a process often referred to as degranulation.1 Early ultrastructural and electrophysiological studies showed that degranulation involves compound exocytosis, which besides the fusion of granules at the PM, implicates fusion between granules, either in a multivesicular or sequential manner, thereby forming degranulation channels3,4 (Fig. 1). At the PM fusion site, the proteoglycan matrix is expelled. Many, but not all,5 of the mediators are tightly bound to this matrix, but are liberated by ion exchange due to the rise in pH and granule swelling.6 The expelled proteoglycan core can sometimes be observed in the tissue surrounding MCs. MCs also exhibit piecemeal degranulation with progressive loss of granule particulate contents without any evidences for fusion.7 Ultrastructural analysis has revealed the budding of small cytoplasmic granules that move towards the PM, leading to gradual emptying.8 Progressive SG emptying could also result from transient fusion, often referred to as “kiss and run” fusion. Such transient fusion events have been shown to occur at a

Figure 1. The different modes of exocytosis in mast cells are illustrated. Generally activation with a strong stimulus induces compound and/or multivesicular exocytosis. With some stimuli another mode of exocytosis called piecemeal degranulation can be observed, which involves gradual emptying of vesicular contents without any observable fusion event.

THE MECHANISMS OF EXOCYTOSIS IN MAST CELLS

109

frequency of approximately 2:1 over full fusion in live imaged MCs.9 MCs can participate in multiple cycles of activation for mediator release. However, in comparison to the very fast synaptic vesicle cycle, the regeneration of a fully reconstituted granular compartment can take up to 72 h.10

CHARAC R TERISTICS OF MAST CELL GRA R NULES A peritoneal MC contains up to 1000 SGs occupying up to 40% of the cell volume.11 While endo- or exocrine cells have specialized SGs, MCs or other cells of hematopoietic origin contain secretory lysosomes, which are organelles containing lysosomal hydrolases situated at the junction between the endo- and exocytic pathways.12 Electron microscopic studies of MCs has revealed granular heterogeneity and allowed to the designation of *          #       <    endocytotic tracers.13 Type I SGs are multivesicular and could represent conventional lysosomes that release their content only under nonphysiological situations. Type II and III SGs could represent the compartment regulated by a physiological stimulus. Type II granules show an electron-dense core surrounded by membrane vesicles and are accessible to endocytotic tracers. Type III granules contain essentially electron-dense material and are not accessible by endocytotic tracers. The biogenetic relationship between these types of SGs remains to be elucidated. It has become clear that the granular compartment     %# {      $  # "   proteoglycans, as genetic targeting of the heparin biosynthesis pathway or the serglycin core in mice revealed severe defects in SG maturation.5

THE BASIC SECRETORY MACHINERY IN MAST CELLS & %*$ ^  MC degranulation is triggered by aggregating IgE bound to Fc¡RI. Recent studies have demonstrated that degranulation relies on the conserved mechanism of membrane fusion, implicating SNARE proteins initially discovered in neurons.2 Each cell type expresses different combinations of SNARE members selectively distributed on intracellular   *# !       <  $ %    with the cell membrane. SNAREs contain an _-helical motif of about 60 aa, called a SNARE motif, four of which combine to form a stable tetrameric core trans-SNARE complex, the formation of which is energetically favored. This promotes vesicle docking with the target membrane and catalyzes fusion of the apposing membranes.14 Functionally, SNAREs      !^~‚=   # !  ^~‚=   # target compartment that can either be the PM or another vesicular compartment. Usually, a v-SNARE consists of a single central arginine-containing SNARE motiff containing vesicle associated membrane protein (VAMP) that pairs with central glutamine-containing t-SNARES, including a member of the synaptosome-associated protein-25 (SNAP-25, (neuronal) or SNAP-23, 29, 47 (ubiquitous) containing two SNARE motifs (light chains) and a Syntaxin subfamily member containing one SNARE motif (heavy chain).15 SNAP-25 subfamily members can be replaced by SNAREs structurally related to the N-terminal half

110

MAST CELL BIOLOGY

of SNAP-25 (as one of the two light chains) and one member of the SNARE subfamily related to the C-terminal half of SNAP-25 (as the other light chain).15 MC exocytosis requires SNAREs, as blockade of their function with NN ethylmaleimide (NEM) strongly impairs degranulation.16 NEM inhibits NN ethylmaleimide-Sensitive Factor (NSF), an ATPase that disassembles SNARE complexes. Transfection of a NSF-mutant unable to promote SNARE disassembly markedly inhibited exocytosis, indicating that SNARE disassembly is an integral part of the cycle that primes SNARE-mediated fusion.17 MCs express a wide range of SNAREs. These include the t-SNAREs SNAP-23 and Syntaxins2, 3, 4 and 6.18-20 Expressed v-SNAREs comprise the VAMP family members VAMP2, 3, 7 and 8.18-20 *#    #  }  $   $ was the PM-localized t-SNARE SNAP-23.18 Introduction of blocking antibodies into permeabilized rat peritoneal MCs (RPMCs) potently inhibited stimulated exocytosis,  $    #$    @20 During exocytosis, SNAP-23 relocated from the PM along degranulation channels that formed in the interior of the cell, explaining a particular function in compound exocytosis.18 Another study showed that overexpression of SNAP-23 in the RBL-2H3 mast cell line enhanced exocytosis.21 Analysis of complex formation in mouse bone marrow derived MCs (BMMCs) and primary human MCs showed that complex formation between SNAP-23 and Syntaxin4 or VAMP8 increased during Fc¡RI-induced stimulation.22 Concerning Syntaxin members, overexpression of the PM-localized Syntaxin4 in RBL-2H3 cells interfered with degranulation.19 Furthermore, introduction of antibodies for Syntaxin4, but not of Syntaxin6, inhibited degranulation in permeabilized cells.20 Like SNAP-23, Syntaxin4 formed enhanced complexes with SNARE partners VAMP8 and SNAP-23 after stimulation.22 In addition, evidence for the implication of Syntaxin3 has been presented. This t-SNARE is primarily located on SGs and relocates to the periphery upon stimulation.17 Furthermore, it is able to form complexes with other SNARE partners involved in exocytosis including SNAP-23 and VAMP7. Preliminary data in our laboratory show that siRN R A-mediated knock-down of Syntaxin3 partly inhibits exocytosis. As Syntaxin3 is located on SGs, it might represent one of the t-SNAREs involved in granule-granule fusion. Concerning VAMP proteins, four studies have implicated VAMP8, a v-SNARE  #%  }         #   between the endocytic and the exocytic apparatus in MCs.16,20,22,23 In several types of MCs, VAMP8 partially colocalizes with the SG markers, serotonin and mMCP-6 (tryptase). Tiwari et al22 #% # †

@   ž \—^   #  $ as monitored by the release of histamine and `-hexosaminidase, but not chemokine/ <  $ *#   ž \— %     !!    VAMP8 markedly inhibited histamine release in passive systemic anaphylaxis experiments. Similarly, introduction of a soluble recombinant VAMP8 protein into RBL-2H3 cells or of blocking antibodies into permeabilized human MCs markedly inhibited release of `-hexosaminidase or histamine.20 # $ $ ž \—^  †

@ $ that VAMP8 may further discriminate between granule subpopulations, as it may control the release of serotonin, cathepsin-D and `-hexosaminidase but not of histamine, which may localize to distinct SGs.23 Distinct release of cathepsin D and histamine containing granules has also been reported when examining the role of syntaptotagminII.24 However, #         # $ #!      and localization of lysosomal and genuine MC granular markers.19,20,22 Similarly, ^  †

@   #% $#    $25 MC activation resulted in the redistribution of VAMP8 from granular compartments to the

THE MECHANISMS OF EXOCYTOSIS IN MAST CELLS

111

PM; and was associated with increased complex formation with PM-membrane localized SNAP-23 and Syntaxin4.20,22 Besides VAMP8, other VAMPs have also been examined.    ž \_  ž \Œ   $  < $  proteins or antibodies, did not affect release, suggesting that they may not play a role.20,22,23 This is consistent with the absence of colocalization with SG markers. However, it was found that ectopically expressed GFP-VAMP2 gradually fused with the PM following stimulation in RBL-2H3 cells.26 Furthermore, a tendency to increased complex formation during stimulation can be observed both for VAMP2 and VAMP3 in the background  ž \—^  $ # #         results were obtained with VAMP7, a lysosomal marker. Initial experiments showed that VAMP7-CFP/YFP colocalizes with Syntaxin3 on SGs and relocates from granular structures to the PM after stimulation in RBL-2H3.27 However, no effect on secretion was seen after introducing soluble VAMP7 into RBL-2H3 cells.16 In human MCs, however, introduction of anti-VAMP7 markedly inhibited histamine release.20 Taken together, these    #   ~‚= $    @  $ *#   SNAP-23 maybe a particular feature of the compound exocytotic process by providing an   ^~‚=     # \ 18 The implication of a second t-SNARE, "Œ          #    $ "

ACCESSORY PROTEINS IN MAST CELL MEMBRA R NE FUSION In vitro reconstitution of the SNARE-mediated fusion process in liposomes exhibits a slow kinetics, suggesting that additional effectors are operative in vivo.28 Besides   #    $ #  $   # $ #  to cell signaling and second messengers. Several SNARE accessory proteins have been described in MCs. NSF and _-SNAP SNARE proteins were initially discovered by their ability to bind to soluble NSF attachment proteins (SNAPs) bridging the interaction to the ATPase NSF,29 which mediates SNARE disassembly, the energy dependent step of fusion. Treatment of MCs with the NSF-inactivating compound NEM dramatically inhibits exocytosis, as does the expression   *\^  ~ $ $  #    # $$  $^  ~‚= "17 *# %    # #    a dominant-negative _-SNAP unable to activate the ATPase.16 These data point out the importance of SNARE disassembly or priming during degranulation. SM Proteins MCs also express SM (Sec1/Munc18) family members that bind Syntaxin proteins    ^    %##   <%   $   ! $  $   < 14 Munc18 (mammalian uncoordinated  unc-18)           # $  " \   in understanding their function has come from knock-out studies of Munc18-1 and Munc18-3 isoforms. They demonstrated an essential role in fusion in various types of regulated-secretion competent cells.14,30,31 The mechanisms could involve distinct

112

MAST CELL BIOLOGY

features that are in part coupled to their different modes of interaction with cognate SNARE proteins.14,32 Binding to the closed “un-complexed” conformation of Syntaxin (containing like the SNARE complex a four-helix bundle composed of three N-terminal helices of the Habc domain and the SNARE motif) blocks SNARE assembly and could play a role in gating the initiation of the fusion reaction.14 Such binding may also have a chaperone-like function, as expression of various Syntaxins was shown to decrease in the absence of Munc18 isoforms.14 By contrast, binding to the trans-SNARE complex may more directly be involved in promoting fusion.33 Evidence obtained from different systems suggest that Munc18 isoforms have additional functions in vesicle translocation, tethering and docking,32 which may not necessarily relate to SNARE binding per se, but could involve interaction with other effectors such as Mints,34 Doc2,35 Granuphilin36 and Cab45.37 MCs express the ubiquitous isoforms Munc18-2 and Munc18-3, while neuronal Munc18-1 has been detected so far only at the mRN R A level.38,39 Functional studies demonstrated a role for Munc18-2, which is localized on SGs and associates to Syntaxin3 and Syntaxin2. Ectopic expression of Munc18-2 and Munc18-2 peptides interfere with an effector loop inhibited MC exocytosis.38 Similarly, siRN R A-mediated knock-down of Munc 18-2 inhibited exocytosis.40 This supports recent data obtained in patients carrying $  $—^_ # #! !     "    of Munc18-2 and a consequent secondary down-regulation of Syntaxin11, causing familial hemophagocytic lymphohistiocytosis Type5 (FHL5) impairing lytic granule exocytosis.41,42 In contrast to other immune cells,43 so far no role has been demonstrated for the PM-localized and Syntaxin4-interacting isoform Munc18-3 in MCs. Munc13-4 Recent studies also indicated a role of the Munc13 (mammalian unc-13) family members in mast cell degranulation.29,44 *# $Œ   #   ^  isoforms, Munc13-1, 13-2 and 13-3; and a nonneuronal isoform Munc13-4, which is expressed in the lung, spleen and hematopoietic cells.44-46 Munc13 family members are ! }    #  !!  !   *#  #  $ constitutively “open” Syntaxin1 can partially rescue the fusion block in Munc13-1/13-2 double KO mice suggests a role in the conformational transition of Syntaxin1.44 Furthermore, the function of Munc13 in priming implies an interaction with the adaptor protein Rim which is able to bind Rab3 family members, both of which play a role in the regulation of fusion.44 Mutations causing a loss of Munc13-4 function expressed in hematopoietic cells impair exocytosis of cytotoxic SGs responsible for familial FHL3.47 In cytotoxic T cells, Munc13-4 has dual function.48 In an early step, it promotes attachment of “exocytotic” vesicles generated from Rab11/Munc13-4-positive and Rab27A-positive endosomal vesicular pools; and then it primes fusion of genuine cytotoxic SGs with these “exocytotic” vesicles to secrete their content. Priming involves interaction of Munc13-4 with Rab27A (see also below). In MCs, both Munc13-4 and Rab27 isoforms colocalize on SGs, where Munc13-4 is anchored via its Munc-homology domain.46 Over-expression of Munc13-4, but not mutated Munc13-4 enhances degranulation, suggesting a positive regulatory function.45,46 The mechanism may involve the interaction with Doc2_, a calcium effector of exocytosis. Munc13-4 and Doc2_ and Rab27A can form a tripartite complex. The importance of Doc2_ is underlined by the marked inhibition of exocytosis in Doc2_^  †

@35

THE MECHANISMS OF EXOCYTOSIS IN MAST CELLS

113

Rab GTPases Rab proteins constitute a large family of small GTPases involved in intracellular   < $       ! $ $  and fusion.49 In particular, Rab3 family members have been recognized as regulators of late steps in exocytosis. At the synapse, they are not essential per se, but function as gatekeepers of calcium-triggered exocytosis by a still poorly understood mechanism.29 Some of their actions could relate to vesicle docking and priming via interaction with effectors such as Rim/Munc13-4 and rabphilin3A. In MCs, Rab3A, Rab3B and Rab3D are expressed when examined by PCR R analysis.50 \  "  %    Rab3A and Rab3D and the latter was shown to translocate from SGs to the PM following stimulation.50,51 Over-expression of Rab3D or a constitutively active GTP bound mutant, but not of Rab3A, revealed an inhibitory effect in antigen-stimulated RBL MCs.50 Another study showed that over-expression of Rab3A also inhibited exocytosis.52 The role of ‚Œ #  # $ @   ‚Œ^   #! "# normal exocytosis and do not reveal any increase in granule size as shown for pancreatic zymogen granules.53 However, the role of compensatory mechanisms by other Rabs has not been addressed. Recently, the implication of another GTPase, Rab27, has been reported.45,54 It exists in two isoforms (Rab27A/Rab27B) known to interact with a variety of Rab27-binding proteins such as Slac2 (synaptotagmin-like protein (Slp) family with tandem C2 @$^ ™  $Œ^’  $      <55 Interaction with Slac2 family members couples Rab27 function to the actin-based motor MyosinVa in melanocytes and neuroendocrine cells.56 This mode of action does not apply  ‚_”^  " *  ˜@*ƒ™ %## ! #   $   granular contents due to a docking defect.47 Mutations in Rab27A are responsible for the FLH2 secretory phenotype in humans. This includes pigmentation defects as, unlike Munc13-4, Rab27A is also expressed in melanocytes. MCs express Rab27A and Rab27B isoforms, which localize to SGs.45,54 However, degranulation defects are only observed  ‚_”†^      $ ‚_”^       \@ were observed in Rab27B and double KO mice, while surprisingly, PCA was enhanced in Rab27A mutant mice.45,54 $ # $  #% # ‚_”†^  MCs exhibit mild SG clustering with a 10-fold increase of SGs in microtubule-dependent movement, suggesting that Rab27B action could regulate the transition from microtubule to actin-based motility. Complexins Recently described SNARE-interacting effectors are the cytosolic proteins, complexins I and II.14 Complexins preferentially bind to assembled SNARE complexes, which then blocks completion of assembly and hence transfer of force required for fusion pore expansion, leading to a “superprimed” state ready for immediate fusion.57 It is thought that # $       $14,44 Calcium binding to the tandem C2A and C2B domain induces simultaneous SNARE and membrane phospholipids binding thereby reversing the complexin block, in addition to the induction of fusion-promoting membrane phospholipids. Complexin may additionally participate in fusion activation through its N-terminal domain binding to a membrane-proximal v-SNARE sequence, thereby pulling the complex closer to the membrane.57

114

MAST CELL BIOLOGY

MCs express Complexin II but not Complexin I.58 Complexin II functions as a positive regulator, as siRN R A-mediated knock-down markedly suppressed IgE-induced degranulation.58 The sensitivity of the degranulation process for calcium was decreased, as knock-down cells required higher extracellular calcium concentrations for exocytosis. ${$  $ #% # $  $ @"    the cytoplasm to the PM, where it might interact with trans-SNARE complexes. Indeed, it binds tightly to a complex composed of Syntaxin3, SNAP-23 and VAMP8 or 2.40 Taken together these studies indicate that Complexin II in MCs, rather than acting as a fusion clamp, may promote fusion by enhancing its calcium sensitivity. Whether this implies concerted action with a synaptotagmin family member remains an open question. Synaptotagmins Synaptotagmins (Syts) comprise a small family of single TM proteins involved in calcium-dependent regulation of exocytosis.29 They contain tandem calcium binding C2 domains (termed C2A and C2B) binding three and two calcium ions, respectively. *#      !    %       by the presence of Syt binding partners. Calcium binding promotes oligomerization and membrane phospholipid binding,44 thereby allowing simultaneous binding to two membranes. It also promotes binding to the SNARE complex, allowing the formation of a quaternary SNARE-synaptotagmin-calcium-phospholipid complex, thereby connecting the basic fusion machinery to calcium-triggered exocytosis.44 MCs express several Syt family members (SytI, II, III and IX).24,59-62 Initial siRN R A <<% $ #% #         # release, but rather blocked release of cathepsin D suggesting that it acts to prevent fusion of genuine lysosomes, but not SGs.24 `%! #  #  #  †

@  %   ^   #%  !    #  `-hexosaminidase and histamine release.25 Unlike in neurons, SytII, which has a perigranular location, does not affect spontaneous granular release. SytIII was found to function as a critical regulator of the perinuclear endocytic recycling compartment (ERC) and could possibly regulate SG size,60 while SytIX functions in protein export from the ERC to the cell surface.61 These data suggest that nonneuronal Syts interfere with distinct steps of     <  @  # ^  " #% SCAMPs A further accessory protein that regulates the fusion competent state are secretory carrier membrane proteins (SCAMPs), a family of ubiquitous membrane proteins of transport vesicles. They are highly expressed in organelles undergoing regulated exocytosis such as synaptic vesicles, but also mast cell SGs.63 They are tetraspanins with the short segment (E peptide) linking the second and third TM domain being the most highly conserved element and unique to SCAMPs.64 MCs express SCAMP1 to SCAMP3 isoforms, with SCAMP1 and SCAMP2 being most prevalent.65 They localize to SGs and vesicles, but a small fraction is also found at the PM colocalizing with Syntaxin4 and SNAP-23. Introduction of the E-peptide potently inhibits exocytosis in permeabilized MCs.65 Further $ #!  #   <  #  $  $‹$  ~\^_Œ relocation and ATP-dependent priming.64,66        @ \_ causes an apparent defect in forming stable fusion pores.63 SCAMPs may act at a step that

THE MECHANISMS OF EXOCYTOSIS IN MAST CELLS

115

couples Arf6-stimulated phospholipase D (PLD) activity to formation of fusion pores, as the peptide also inhibits PLD activity.64 Furthermore, in PC12 cells it has been shown that a SCAMP2 mutant with decreased Arf6-binding recapitulated the inhibitory effect. This could be in agreement with the proposed implication of PLD in MC exocytosis.67,68 *   ;>   MC degranulation involves extensive cytoskeletal reorganization with dissolution  # $  ^ "     ^ ${69 In parallel, microtubules stretch out into the lamellipodia formed during the stimulation process.70 $ #          $ % <  <  $$  with hook-like structures.71 The cytoskeleton could provide an appropriate scaffold for signaling proteins and the attachment of cellular compartments and proteins of the fusion machinery. The actino-myosin contractile system or tubulin-based motors could be important for the mobilization and extrusion of SG content.72 On the other hand, cortical actin could represent a substantial barrier. Convincing evidence for the requirement of cortical actin depolymerization has come from studies combining atomic force and confocal microscopy, which showed that granule fusion occurs only at PM sites devoid of cortical actin.73 Similarly, treatment with actin-depolymerizing drugs or ectopic expression of a constitutively active mutant of Rho R A, Rac1 or CDC42 promoting actin remodeling enhances secretion, while dominant-negative forms inhibited secretion.69,74 However, other functional consequences of these treatments, notably interference with early cellular signaling pathways, have been described, suggesting a complex relationship.74,75 Concerning the tubulin network, treatment with microtubule destabilizing or stabilizing drugs affected secretion in MCs.38,76  $ @     

$—^_^ %  "$   ^^ ${ $     aligned along newly formed microtubular tracks, suggesting microtubular transport mechanisms.38 In agreement, live imaging of SGs revealed bidirectional movement in both resting and activated cells adjacent to microtubules; and the rate and extent of exocytosis was inhibited by the microtubular depolymerizing drug Colchicine.77 A recent study showed that degranulation can be separated into two independent pathways based on the following: i) the microtubule-dependent and Fyn/Gab2/Rho R A-regulated translocation of CD63-GFP-positive SGs to the PM occurred in a calcium-independent manner; and ii) the actual fusion required increases in intracellular calcium concentrations.76 Thus, it is possible that, similar to other hematopoietic cells with a more locally restricted release,78 the microtubular system also plays an important role in the secretory process of MC. These  #%!    %# $ %##   !    ! $  exocytosis in RBL cells.9 Similarly, RPMCs with a highly differentiated SG compartment rather show an inward movement of PM components such as SNAP-23.18 Still, the absence of movement does not exclude a coordinated action between the cytoskeleton and the fusion apparatus, for example, to guide the extensive membrane rearrangements and to provide pulling force. *< #         $     #!   # implication of cytoskeletal elements in the late steps of secretion. This is largely due to the complexity of the molecular pathways involved, which not only impinge on the late steps of exocytosis, but on a whole variety of cellular processes. Nevertheless, accumulated data suggests that cytoskeletal functions are an integral part of the fusion process in MCs.

116

MAST CELL BIOLOGY

COUPLING OF THE FUSION MACHINERY TO SIGNAL TRA R NSDUCTION FOR EXOCYTOSIS IN MAST CELLS & *  \   Aggregation of Fc¡RI is a potent stimulus for degranulation. This allows docking of nonreceptor tyrosine kinases, thus launching an activation cascade.1 Protein phosphorylation-dephosphorylation represents an important regulatory mechanism in the late steps of MC exocytosis. Several fusion proteins have been shown to become phosphorylated by Ser-Thr kinases, including PKC, CaM kinaseII, PKA, Casein kinaseII. Phosphorylation could play a variety of functions in the fusion process including regulation of the activity state of SNARE proteins79 or a direct interference in the fusion process.80 In MCs, activation of protein kinaseC (PKC) represents an essential signal for secretion. A calcium-dependent isoform, PKC` and a calcium-independent isoform, PKCb, have been implicated using depletion-reconstitution studies.81 The role of PKC` %    \@`^  @82 while PKCb^  @ $ #% enhanced exocytosis.83 These apparent differences may be explained by indirect effects of PKCb on negative regulatory mechanisms. Regardless, PKC clearly regulates late steps in exocytosis. Molecular targets include myosin light and heavy chains necessary for the reorganization of the actino-myosin cortex during secretion.84 PKC also phosphorylates Munc18-1 within its second domain, which prevents Syntaxin binding.85 Similarly, ##   $—^Œ  \@ $     "_  ’  platelets86 and thus could regulate the amount of available fusion-competent Syntaxin. PKC also phosphorylates SNAP-25 and Syntaxin4 in vitro.87 In MCs, about 10% of SNAP-23 becomes transiently phosphorylated during degranulation on Ser95/Ser120 within its cysteine-rich linker region.88 While PKC was a possible effector, recent data have implicated IgB kinase (IKK), generally known to phosphorylate IgB, which induces nuclear translocation of the NF-gB transcription factor.89 ^  @ #!  <    $  #  %# $  @^  mice. This was independent of NF-gB translocation. In vivo, IKK K phosphorylated residues Ser95/Ser120  $ %# ## ^ $  ^  MCs partially restored, while over-expression of phosphorylation mutants inhibited IgE-stimulated exocytosis.88,89 #  #% #  ^   SNAP-23 formed fewer complexes with Syntaxin4, VAMP2 and VAMP8, suggesting that phosphorylation on Ser95/Ser120 is required for complex formation. A different calcium-regulated kinase activity present in Rab3D-containing $  %     ##   "’    to SNAP-23, the phosphorylation of Syntaxin4 decreased its SNAP-23 binding capacity, although so far this effect has only been studied in vitro.79 Besides PKC, SNARE Kinase (SNAK) could also be a regulator of exocytosis in MCs.90 SNAK K phosphorylates SNAP-23 at Ser-Thr residues thereby increasing the stability of newly synthesized protein. SNAK-dependent phosphorylation could thus indirectly promote SNARE complex formation by increasing the pool of available SNAP-23. Protein phosphorylation is regulated by a dynamical balance between the action of kinases and phosphatases. Phosphatases likely also regulate fusion in MCs.91 Inhibitors of phosphatase PP1 and PP2a reduced secretion even when bypassing early-receptor-mediated signals.92 PP2A is recruited to the PM in a manner that correlates with the kinetics of secretion. Subsequent studies in RBL-2H3 cells have demonstrated that both PP1 and

THE MECHANISMS OF EXOCYTOSIS IN MAST CELLS

117

PP2A transiently associate with cortical myosinII, suggesting a role in the cytoskeletal rearrangements of the actino-myosin cortex.93 A crucial target for phosphatase activity could be the PKC-dependent phosphorylation sites in myosin light and heavy chains. Another phosphatase implicated in membrane fusion includes megakaryocyte cytosolic protein tyrosine phosphatase 2 (MEG2) localized to SG, whose over-expression results in the formation of large granules.94 MEG2 is known to dephosphorylate NSF on a key tyrosine residue important for its activity.95

TARGETS OF CALCIUM Although it was realized more than 50 years ago that calcium is an essential intracellular messenger for secretion in neurons and somewhat later in MC,96 the molecular targets remained obscure for many years. The synaptotagmin calcium sensors have now emerged as prime candidates. They contain the conserved tandem calcium and phospholipid-binding C2 domains (C2A and C2B).44 Calcium binding promotes the     $^$ " #$#     # %# ~‚=   " #  $ Another calcium sensor is calmodulin (CaM) interacting with numerous proteins that have a role in secretion.97 Results of inhibitor studies in MCs range from no effect to an #     –     $     %#   # $  this sensor.98,99 In MCs, potential targets include myosin light chain kinase (MLCK),100 CaM kinaseII which phosphorylates nonmuscle myosin,101 Syntaxin3102 and PLD.67 CaM binds also VAMP2.103 CaM is thought to activate VAMP2 by liberating it from its lipid-bound state upon binding.103 In agreement, microinjection of a VAMP2-derived peptide that < @ ^  #    $  #  "103 However, calmodulin-binding has not been observed for VAMP8, which has been described as a major v-SNARE in MC exocytosis. In addition, a variety of other molecules that coordinate the secretory fusion process are also calcium sensors. These include Munc13-4 and Doc2_, which are also known to express calcium-binding C2 domains.44 Evidence for Lipid Raft Domains in Mast Cell Exocytosis Lipid rafts are dynamic assemblies of cholesterol and sphingolipids, which form a separate liquid-ordered phase in membranes that are enriched in discrete subsets of proteins. At the PM, they regulate signal transduction by providing concentrated platforms for signaling proteins.104 Lipid rafts are also found in biosynthetic and endocytic pathways.105 Raft-dependent interactions may also play a role in exocytosis, as treatment with cholesterol-depleting agents considerably affected regulated secretion in several cellular systems.106 Moreover, both vesicular and PM fusion proteins become enriched in lipid rafts, although the nature of this compartment is still somewhat under dispute.107,108      <  $  #   # ^ $  *#$ similar to the platforms for transmembrane signaling, such domains may also form functional entities for fusion. In MCs and when examined by sucrose gradient fractionation, SNAREs were either excluded (Syntaxin2), equally distributed between raft and nonraft fractions (Syntaxin4, VAMP8, VAMP2), or selectively enriched in rafts (Syntaxin3, SNAP-23).109 The accessory protein Munc18-2 and 18-3 were found in nonraft fractions.

118

MAST CELL BIOLOGY

However, small amounts of Munc18-2 also consistently distributed into rafts. Cognate SNARE complexes of Syntaxin3 with SNAP-23 or VAMP8 were also enriched in rafts, while those of Syntaxin3 with Munc18-2 were excluded. A separate study110 also found a selective enrichment in raft fractions of SNAP-23/Syntaxin4 and Syntaxin4/VAMP2 complexes. In addition, quantitative analysis showed that raft-associated Syntaxin4 and VAMP2 increased about twofold in Fc¡RI-stimulated cells, forming complexes containing Syntaxin4, VAMP2 and phospho-SNAP-23. Taken together, these data point out an important role of the membrane lipid environment in MC exocytosis.

CONCLUSION MC-triggered compound exocytosis is fundamental in immunity, but also plays a     {   ƒ< #     @ $   and highly regulated, secretory machinery that includes cognate SNARE membrane fusion proteins as well as various accessory regulatory proteins. These proteins and the evidence for implication are summarized in Figure 2. Together, this machinery

Figure 2. $ #  "      $     ## regulated secretory machinery that includes cognate SNARE membrane fusion proteins such as the vesicle-associated membrane proteins (VAMP7/8), the plasma membrane-associated syntaxin4 (eventually Syntaxin3 in case of granule-granule fusion) and SNAP-23. These enable formation of a stable multi-molecular complex that energetically favors the merger of lipid membranes. SNARE complex formation is regulated by multiple accessory proteins that may either directly assist the fusion         !     #     $  !  cytoskeletal rearrangement. For detailed explanation of their mechanism refer to the text. Note that fusion at the PM occurs preferentially in lipid rafts.

THE MECHANISMS OF EXOCYTOSIS IN MAST CELLS

119

coordinately functions to protect the organism from unwanted release of potentially dangerous substances; but also promotes release upon arrival of a physiologic signal at the surface. In disease, besides inappropriate triggering, MCs often show a hyper-activated phenotype, translating into inappropriate regulation. Further research efforts will continue to focus on the molecular understanding of this machinery.

ACKNOWLEDGEMENTS The work of Ulrich Blank is supported by Inserm, CNRS and Université Paris 7 as well as a grant by the French National Research Agency, under reference A R-08-GENO-025-01. AN

REFERENCES 1. Blank U, Rivera J. The ins and outs of IgE-dependent mast-cell exocytosis. Trends Immunol 2004; 25(5):266-73. 2. Südhof T. The synaptic vesicule cycle: a cascade of protein-protein interactions. Nature 1995; 375:645-53. 3. Alvarez de Toledo G, Fernandez J. Compound versus multigranular exocytosis in peritoneal cells. J Gen Physiol 1990; 95:397-402. ’ ‚## \  ^    #     #    =" @ ‚ 1975; 93(2):293-8. 5. Pejler G, Abrink M, Ringvall M et al. Mast cell proteases. Adv Immunol 2007; 95:167-255. 6. Monck JR, Oberhauser AF, Alvarez de Toledo G et al. Is swelling of the secretory granule matrix the force that dilates the exocytotic fusion pore? Biophys J 1991; 59(1):39-47. 7. Crivellato E, Nico B, Mallardi F et al. Piecemeal degranulation as a general secretory mechanism? Anat Rec A Discov Mol Cell Evol Biol 2003; 274(1):778-84. 8. Dvorak AM, Hammond ME, Morgan E et al. Evidence for a vesicular transport mechanism in guinea pig basophilic leukocytes. Lab Invest 1980; 42(2):263-76. 9. Williams RM, Webb WW. Single granule pH cycling in antigen-induced mast cell secretion [In Process Citation]. J Cell Sci 2000; 113(Pt 21):3839-50. 10. Galli S, Dvorak A, Dvorak H. Basophils and Mast Cells: Morphologic Insights into their Biology, Secretory Patterns and Function. Prog Allergy 1984; 34:1-141. 11. Alvarez de Toledo G, Fernandez JM. Patch-clamp measurements reveal multimodal distribution of granule sizes in rat mast cells. J Cell Biol 1990; 110(4):1033-9. _  #     ¢  #  $    #  *  Cell Biology 1996; 6:329-32. 13. Raposo G, Tenza D, Mecheri S et al. Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation. Mol Biol Cell 1997; 8(12):2631-45. 14. Sudhof TC, Rothman JE. Membrane fusion: grappling with SNARE and SM proteins. Science 2009; 323(5913):474-7.  ` & ~‚=     †# †#  _••– ”’’˜_™_•^’’ 16. Lippert U, Ferrari DM, Jahn R. Endobrevin/VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells. FEBS Lett 2007; 581(18):3479-84. 17. Puri N, K Kruhlak MJ, Whiteheart SW et al. Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly. J Immunol 2003; 171(10):5345-52. 18. Guo Z, Turner C, Castle D. Relocation of the t-SNARE SNAP-23 from lamellipodia-like cell surface projections regulates compound exocytosis in mast cells. Cell 1998; 94(4):537-48. 19. Paumet F, Le Mao J, Martin S et al. Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3   $   " ’  "      !^   protein 8-containing secretory compartment. J Immunol 2000;164(11):5850-7. 20. Sander LE, Frank SP, Bolat S et al. Vesicle associated membrane protein (VAMP)-7 and VAMP-8, but not VAMP-2 or VAMP-3, are required for activation-induced degranulation of mature human mast cells. Eur J Immunol 2008; 38(3):855-63. 21. Vaidyanathan VV, Puri N, Roche PA. The last exon of SNAP-23 regulates granule exocytosis from mast cells. J Biol Chem 2001; 276(27):25101-6.

120

MAST CELL BIOLOGY

22. Tiwari N, Wang CC, Brochetta C et al. VAMP-8 segregates mast cell-preformed mediator exocytosis from <   < #% † _••—– ˜”™Œ““^”’ 23. Puri N, Roche PA. Mast cells possess distinct secretory granule subsets whose exocytosis is regulated by different SNARE isoforms. Proc Natl Acad Sci USA 2008; 105(7):2580-5. 24. Baram D, Adachi R, Medalia O et al. Synaptotagmin II negatively regulates Ca2 -triggered exocytosis of lysosomes in mast cells. J Exp Med 1999; 189(10):1649-58. 25. Tuvim MJ, Mospan AR, Burns KA et al. Synaptotagmin 2 couples mucin granule exocytosis to Ca2

signaling from endoplasmic reticulum. J Biol Chem 2009; 284(15):9781-7. 26. Miesenbock G, De Angelis DA, Rothman JE. Visualizing secretion and synaptic transmission with `^!   {$    ~$  €€—– Œ€’˜““—€™€_^ 27. Hibi T, Hirashima N, Nakanishi M. Rat basophilic leukemia cells express syntaxin-3 and VAMP-7 in granule membranes. Biochem Biophys Res Commun 2000; 271(1):36-41. 28. Weber T, Zemelman BV, McNew JA et al. Snarepins—minimal machinery for membrane fusion. Cell 1998; 92(6):759-72. 29. Lang T, Jahn R. Core proteins of the secretory machinery. Handb Exp Pharmacol 2008; (184):107-27. 30. Kanda H, Tamori Y, Shinoda H et al. Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization. J Clin Invest 2005; 115(2):291-301. 31. Oh E, Spurlin BA, Pessin JE et al. Munc18c heterozygous knockout mice display increased susceptibility for severe glucose intolerance. Diabetes 2005; 54(3):638-47. 32. Burgoyne RD, Barclay JW, Ciufo LF et al. The functions of Munc18-1 in regulated exocytosis. Ann N Y Acad Sci 2009; 1152:76-86. 33. Shen J, Tareste DC, Paumet F et al. Selective activation of cognate SNAREpins by Sec1/Munc18 proteins. Cell 2007; 128(1):183-95. 34. Ho A, Morishita W, Atasoy D et al. Genetic analysis of Mint/X11 proteins: essential presynaptic functions of a neuronal adaptor protein family. J Neurosci 2006; 26(50):13089-101. 35. Higashio H, Nishimura N, Ishizaki H et al. Doc2 alpha and Munc13-4 regulate Ca(2 ) -dependent secretory lysosome exocytosis in mast cells. J Immunol 2008; 180(7):4774-84. 36. Fukuda M, Imai A, Nashida T et al. Slp4-a/granuphilin-a interacts with syntaxin-2/3 in a Munc18-2-dependent manner. J Biol Chem 2005; 280(47):39175-84. 37. Lam PP, Hyvarinen K, Kauppi M et al. A cytosolic splice variant of Cab45 interacts with Munc18b and impacts on amylase secretion by pancreatic acini. Mol Biol Cell 2007; 18(7):2473-80. 38. Martin-Verdeaux S, Pombo I, Iannascoli B et al. Analysis of Munc18-2 compartmentation in mast cells reveals a role for microtubules in granule exocytosis. J Cell Sci 2003; 116:325-34. 39. Nigam R, Sepulveda J, Tuvim M et al. Expression and transcriptional regulation of Munc18 isoforms in mast cells. Biochim Biophys Acta 2005; 1728(1-2):77-83. 40. Tadokoro S, Kurimoto K T, Nakanishi M et al. Munc18-2 regulates exocytotic membrane fusion positively interacting with syntaxin-3 in RBL-2H3 cells. Mol Immunol 2007; 44(13):3427-33. ’ @  

 †$     $—^_   $  ## lymphohistiocytosis type 5 and impairs cytotoxic granule exocytosis in patient NK K cells. J Clin Invest 2009. 42. zur Stadt U, Rohr J, Seifert W et al. Familial hemophagocytic lymphohistiocytosis type 5 (FHL-5) is caused by mutations in Munc18-2 and impaired binding to syntaxin 11. Am J Hum Genet 2009; 85(4):482-92. 43. Brochetta C, Vita F, Tiwari N et al. Involvement of Munc18 isoforms in the regulation of granule exocytosis in neutrophils. Biochim Biophys Acta 2008; 1783(10):1781-91. 44. Rizo J, Rosenmund C. Synaptic vesicle fusion. Nat Struct Mol Biol 2008; 15(7):665-74. 45. Goishi K, Mizuno K, Nakanishi H et al. Involvement of Rab27 in antigen-induced histamine release from rat basophilic leukemia 2H3 cells. Biochem Biophys Res Commun 2004; 324(1):294-301. 46. Neeft M, Wieffer M, de Jong AS et al. Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells. Mol Biol Cell 2005; 16(2):731-41. 47. Feldmann J, Callebaut I, Raposo G et al. Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3). Cell 2003; 115(4):461-73. 48. Menager MM, Menasche G, Romao M et al. Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4. Nat Immunol 2007; 8(3):257-67. ’€  < ` ‚ *\      !    ~ ‚!  @ † _••€– •˜—™Œ^_ 50. Roa M, Paumet F, Lemao J et al. Involvement of the ras-like GTPase rab3d in RBL-2H3 mast cell exocytosis % $ ! ##   =   ˜  ‚™ > $ €€”– €˜“™_—^_Œ 51. Tuvim MJ, Adachi R, Chocano JF et al. Rab3D, a small GTPase, is localized on mast cell secretory granules and translocates to the plasma membrane upon exocytosis. Am J Respir Cell Mol Biol 1999; 20(1):79-89. 52. Smith J, Thompson N, Armstrong J et al. Rat Basophilic Leukaemia (RBL) cells overexpressing rab3a have a reversible block in antigen-stimulated exocytosis. Biochem J 1997; 323:321-8. 53. Riedel D, Antonin W, Fernandez-Chacon R et al. Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules. Mol Cell Biol 2002; 22(18):6487-97.

THE MECHANISMS OF EXOCYTOSIS IN MAST CELLS

121

54. Mizuno K, Tolmachova T, Ushakov DS et al. Rab27b regulates mast cell granule dynamics and secretion. *   _••”– —˜”™——Œ^€_  $<$  ž  ‚  ‚_”    *  < $  # ‚_” =  J Biochem (Tokyo) 2005; 137(1):9-16. “ &$  ‚   # `           ^ž ~ @ † _••_– 4(4):271-8. 57. Maximov A, Tang J, Yang X et al. Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 2009; 323(5913):516-21. 58. Tadokoro S, Nakanishi M, Hirashima N. Complexin II facilitates exocytotic release in mast cells by enhancing Ca2 sensitivity of the fusion process. J Cell Sci 2005; 118(Pt 10):2239-46. 59. Baram D, Mekori YA, Sagi-Eisenberg R. Synaptotagmin regulates mast cell functions. Immunol Rev 2001; 179:25-34. 60. Grimberg E, Peng Z, Hammel I et al. Synaptotagmin III is a critical factor for the formation of the perinuclear endocytic recycling compartment and determination of secretory granules size. J Cell Sci 2003; 116(Pt 1):145-54. 61. Haberman Y, Grimberg E, Fukuda M et al. Synaptotagmin IX, a possible linker between the perinuclear endocytic recycling compartment and the microtubules. J Cell Sci 2003; 116(Pt 21):4307-18. 62. Haberman Y, Ziv I, Gorzalczany Y et al. Classical protein kinase C(s) regulates targeting of synaptotagmin IX to the endocytic recycling compartment. J Cell Sci 2005; 118(Pt 8):1641-9. 63. Fernandez-Chacon R, de Toledo GA, Hammer RE et al. Analysis of SCAMP1 function in secretory vesicle exocytosis by means of gene targeting in mice. J Biol Chem 1999; 274(46):32551-4. 64. Castle JD, Guo Z, Liu L. Function of the t-SNARE SNAP-23 and secretory carrier membrane proteins (SCAMPs) in exocytosis in mast cells. Mol Immunol 2002; 38(16-18):1337-40. “ $   ƒ$ ƒ @     \ $    !     $ "        membrane protein (SCAMP2)-derived peptide. J Biol Chem 2002; 277(38):35357-63. 66. Liu L, Guo Z, Tieu Q et al. Role of secretory carrier membrane protein SCAMP2 in granule exocytosis. Mol Biol Cell 2002; 13(12):4266-78. 67. Peng Z, Beaven MA. An essential role for phospholipase D in the activation of protein kinase C and degranulation in mast cells. J Immunol 2005; 174(9):5201-8. 68. Ichikawa S, Walde P. Phospholipase D-mediated aggregation, fusion and precipitation of phospholipid vesicles. Langmuir 2004; 20(3):941-9. 69. Koffer A, Tatham P, Gomperts B. Changes in the state of Actin during the exocytotic reaction of permeabilized rat mast cell. The Journal of Cell Biology 1990; 111:919-27. 70. Oliver C, Sahara N, Kitani S et al. Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation. J Cell Biol 1992; 116(3):635-46. ” ~ =`  $ % < $ $     $     > @  €€•– €“ (Pt 1):43-6. 72. Pendleton A, Koffer A. Effects of latrunculin reveal requirements for the actin cytoskeleton during secretion from mast cells. Cell Motil Cytoskeleton 2001; 48(1):37-51. 73. Deng Z, Zink T, Chen HY et al. Impact of actin rearrangement and degranulation on the membrane structure of primary mast cells: a combined atomic force and laser scanning confocal microscopy investigation. Biophys J 2009; 96(4):1629-39. 74. Norman J, Price L, Ridley A et al. The small GTP-binding proteins, Rac and R Rho, regulate cytoskeletal organization and exocytosis in mast cells by parallel pathways. Mol Biol Cell 1996; 7:1429-42. 75. Hong-Geller E, Holowka D, Siraganian RP et al. Activated Cdc42/Rac reconstitutes Fcvarepsilon RI-mediated Ca2 mobilization and degranulation in mutant RBL mast cells. Proc Natl Acad Sci USA 2001; 98(3):1154-9. 76. Nishida K, Yamasaki S, Ito Y et al. Fc{epsilon}RI-mediated mast cell degranulation requires calcium-independent microtubule-dependent translocation of granules to the plasma membrane. J Cell Biol 2005; 170(1):115-26. 77. Smith AJ, Pfeiffer JR, Zhang J et al. Microtubule-dependent transport of secretory vesicles in RBL-2H3  *   _••Œ– ’˜™Œ•_^_ 78. Clark RH, Stinchcombe JC, Day A et al. Adaptor protein 3-dependent microtubule-mediated movement of lytic granules to the immunological synapse. Nat Immunol 2003; 4(11):1111-20. 79. Pombo I, Martin-Verdeaux S, Iannascoli B etal. IgE receptor type I-dependent regulation of a Rab3D-associated kinase. A possible link in the calcium-dependent assembly of SNARE complexes. J Biol Chem 2001; 12:12. 80. Turner KM, Burgoyne RD, Morgan A. Protein phosphorylation and the regulation of synaptic membrane    *  ~$  €€€– __˜•™’€^“’ 81. Ozawa K, Yamada K, Kazanietz MG et al. Different isozymes of protein kinase C mediate feedback inhibition of phospholipase C and stimulatory signals for exocytosis in rat RBL-2H3 cells. J Biol Chem 1993; 268(4):2280-3.

122

MAST CELL BIOLOGY

82. Nechushtan H, Leitges M, Cohen C et al. Inhibition of degranulation and interleukin-6 production in mast   !         < @ † _•••– €˜™”_^” 83. Leitges M, Gimborn K, Elis W et al. Protein kinase C-delta is a negative regulator of antigen-induced mast cell degranulation. Mol Cell Biol 2002; 22(12):3970-80. 84. Ludowyke RI, Scurr LL, McNally CM. Calcium ionophore-induced secretion from mast cells correlates with myosin light chain phosphorylation by protein kinase C. J Immunol 1996; 157(11):5130-8. 85. Fujita Y, Sasaki T, Fukui K et al. Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein Kinase C. The Journal of Biological Chemistry 1996; 271:7265-8. 86. Reed GL, Houng AK, Fitzgerald ML. Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is phosphorylated after thrombin activation: implications for platelet secretion. Blood 1999; 93(8):2617-26. 87. Chung SH, Polgar J, Reed GL. Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets. J Biol Chem 2000; 275(33):25286-91. 88. Hepp R, Puri N, Hohenstein AC et al. Phosphorylation of SNAP-23 regulates exocytosis from mast cells. J Biol Chem 2005; 280(8):6610-20. 89. Suzuki K, Verma IM. Phosphorylation of SNAP-23 by IkappaB kinase 2 regulates mast cell degranulation. Cell 2008; 134(3):485-95. 90. Cabaniols JP, Ravichandran V, Roche PA. Phosphorylation of SNAP-23 by the novel kinase SNAK K regulates t-SNARE complex assembly. Mol Biol Cell 1999; 10(12):4033-41. 91. Marash M, Gerst JE. Phosphorylation of the autoinhibitory domain of the Sso t-SNAREs promotes binding of the Vsm1 SNARE regulator in yeast. Mol Biol Cell 2003; 14(8):3114-25. 92. Ludowyke RI, Holst J, Mudge LM et al. Transient translocation and activation of protein phosphatase 2A during mast cell secretion. J Biol Chem 2000; 275(9):6144-52. 93. Holst J, Sim AT, Ludowyke RI. Protein phosphatases 1 and 2A transiently associate with myosin during the peak rate of secretion from mast cells. Mol Biol Cell 2002; 13(3):1083-98. 94. Wang X, Huynh H, Gjorlofff Wingren A et al. Enlargement of secretory vesicles by protein tyrosine phosphatase PTP-MEG2 in rat basophilic leukemia mast cells and Jurkat J T-cells. J Immunol 2002; 168(9):4612-9. 95. Huynh H, Bottini N, Williams S et al. Control of vesicle fusion by a tyrosine phosphatase. Nat Cell Biol 2004; 6(9):831-9. 96. Foreman JC, Mongar JL, Gomperts BD. Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretory process. Nature 1973; 245(5423):249-51. 97. Chin D, Means AR. Calmodulin: a prototypical calcium sensor. Trends Cell Biol 2000; 10(8):322-8. 98. Peachell PT, Pearce FL. Effect of calmodulin inhibitors on histamine secretion from mast cells. Agents Actions 1985; 16(1-2):43-4. 99. Sullivan R, Burnham M, Torok K et al. Calmodulin regulates the disassembly of cortical F-actin in mast cells but is not required for secretion. Cell Calcium 2000; 28(1):33-46. 100. Choi OH, Adelstein RS, Beaven MA. Secretion from rat basophilic RBL-2H3 cells is associated with diphosphorylation of myosin light chains by myosin light chain kinase as well as phosphorylation by protein kinase C. J Biol Chem 1994; 269(1):536-41. 101. Buxton DB, Adelstein RS. Calcium-dependent threonine phosphorylation of nonmuscle myosin in stimulated RBL-2H3 mast cells. J Biol Chem 2000; 275(44):34772-9. 102. Risinger C, Bennett MK. Differential phosphorylation of syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) isoforms. Journal of Neurochemistry 1999; 72(2):614-24. 103. Quetglas S, Iborra C, Sasakawa N et al. Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis. EMBO J 2002; 21(15):3970-9. 104. Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 2000; 1(1):31-9. • $<# ‘  "  ‚ ‚     }          < *   _•••– ˜Œ™_•Œ^ •“ ƒ  ž < \  *   ‚   ~\        <  Madin-Darby canine kidney cells. Proc Natl Acad Sci USA 1999; 96(7):3734-8. •” ƒ * † $  &}    ~‚=      # ^ $  #   docking and fusion sites for exocytosis. EMBO J 2001; 20(9):2202-13. 108. Chamberlain LH, Burgoyne RD, Gould GW. SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis. Proc Natl Acad Sci USA 2001; 98(10):5619-24. 109. Pombo I, Rivera J, Blank U. Munc18-2/syntaxin3 complexes are spatially separated from syntaxin3-containing SNARE complexes. FEBS Lett 2003; 550(1-3):144-8. 110. Puri N, Roche PA. Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis. *   _••“– ”˜™’—_^€’

CHAPTER R8 AN EMERGING ROLE FOR THE LIPID MEDIATOR SPHINGOSINE-1-PHOSPHATE IN MAST CELL EFFECTOR R FUNCTION AND ALLERGIC DISEASE Ana Olivera and Juan Rivera Laboratory of Molecular Immunogenetics, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, USA     

=   

Abstract:

Sphingosine-1-phosphate (S1P) plays important roles regulating functions of diverse biological systems, including the immune system. S1P affects immune cell function mostly by acting through its receptors at the cell membrane but it can also induce S1P receptor-independent responses in the cells where it is generated. S1P produced in allergically-stimulated mast cells mediates degranulation, cytokine and lipid mediator production and migration of mast cells towards antigen by mechanisms that are both S1P receptor-dependent and independent. Even in the absence of an antigen challenge, the differentiation and responsiveness of mast cells can be affected by chronic exposure to elevated S1P from a nonmast cell source, which may occur under pathophysiological conditions, potentially leading to the hyper-responsiveness of mast cells. The role of S1P extends beyond the regulation of the function of mast cells to the regulation of the surrounding or distal environment. S1P is exported out of antigen-stimulated mast cells and into the " $   # $ \   %# # $  {$ diverse surrounding tissue cells and several aspects of the allergic disease, such  {  $  $ #      # vasoactive mediators released systemically by mast cells induce the production of S1P in nonhematopoietic compartments, where it plays a role in regulating the vascular tone and reducing the hypotension characteristic of the anaphylactic shock and thus helping the recovery. The dual actions of S1P, promoting the immediate response of mast cells, while controlling the systemic consequences of mast cell activity will be discussed in detail.

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 123

124

MAST CELL BIOLOGY

INTRODUCTION Sphingosine-1-phosphate (S1P) is a lipid mediator, formed by phosphorylation of sphingosine (SPH), that is involved in multiple physiological and pathological processes. Of the vast numbers of processes known to be regulated by S1P, vascular development, !$    $   #   < #!  extensively explored in the last decade.1-4 The involvement of S1P in these physiological and pathophysiological processes results from its ability to modulate important cellular events such as chemotaxis, cytoskeletal changes, survival and proliferation.5,6 In the last few years, it has become clear that the involvement of S1P in immunological responses       $  #   < $ "  # $ of immune cell function.7 In mast cells, S1P regulates chemotaxis and mast cell effector responses such as degranulation, cytokine and lipid mediator production.8 A novel picture of S1P as functioning beyond its role as a regulator of mast cell function in allergic disease is emerging, with the new discovery of its role in termination of some allergic reactions.9

S1P AND ITS TARGETS The generation of S1P is mediated by the enzymatic activity of two cytosolic sphingosine kinase isoforms (SphK1 and SphK2) and occurs mostly at the membrane where the substrate resides and to where these enzymes are translocated after activation.6,10,11 S1P has enough aqueous solubility to partition in the soluble fraction and to diffuse between intracellular membranes.5 However, the site of action is not merely intracellular.6 S1P   "  $   !              ! G-protein coupled receptors (S1PR1-5) present in the cells of origin or in neighboring     #%    # $    isoforms of heterotrimeric GTP-binding proteins.5,6,12,13 Thus, engagement of any receptor that promotes activation of SphKs and generation of S1P can potentially cause trans-activation of S1P receptors, enhancing the signaling repertoire and creating another wave of signals. Beyond its effect on S1PRs, the acute generation of S1P can also affect certain cell functions independently of its receptors6,10 by either binding and modifying putative intracellular targets or by affecting the relative levels of other lipid products, particularly SPH and ceramide whose effects generally oppose those of S1P1,5,6 (Fig. 1). ‚ #  %    #  <%  $    \ S1P generated in the nucleus, via the translocation and activation of SphK2, was found to bind histone deacetylase, inhibiting its activity and thus enhancing gene transcription.14

REGULATION AND FUNCTION OF S1P IN TISSUES AND CIRCULATORY FLUIDS The levels of S1P inside cells and the interstitium of tissues are usually low in homeostatic conditions, due to irreversible degradation of S1P by a S1P lyase15,16 and to its ##   \`  %   \ ## ˜\\  \\_™17 (Fig. 1) present in intracellular membranes and/or at the plasma membrane facing the extracellular environment. However, local increases in S1P levels may occur under certain conditions,

S1P IN MAST CELL EFFECTOR FUNCTION AND ALLERGIC DISEASE

125

Figure 1. Sphingolipid metabolism and biological functions of sphingolipid derived molecules. Ceramide, sphingosine and sphingosine-1-phosphate are lipid mediators derived from complex sphingolipids by the action of several enzymes acting on cellular membranes. Sphingomyelinases cleave the phosphocholine group of sphingomyelin to yield ceramide; ceramidases then cleave ceramide to form sphingosine and sphingosine kinases phosphorylate sphingosine to form sphingosine-1-phosphate (S1P). These enzymes can be selectively or sequentially activated by various stimuli. The sphingolipid metabolites are interconvertible: S1P can be dephosphorylated to form sphingosine by S1P phosphatases and sphingosine acetylated by ceramide synthase. S1P can also be irreversibly degraded by a S1P lyase and the products of its activity used for the resynthesis of lipids. While ceramide and sphingosine were shown to induce apoptosis and cell cycle arrest, S1P prevents apoptosis and promotes cell growth. S1P mediates numerous $        $      ! ^  G-protein-coupled receptors (known as S1PR1–S1PR5) that are differentially expressed by most cells   $ $ \  $  $    "  $      transporters, or to a lesser extent, it may be generated in the extracellular space.

inducing responses in tissue resident or circulating immune cells (which express various combinations of S1PRs) affecting physiological processes.7 For example, during the     {    \  #   {  ! # S1PR1 in T cells18 and hematopoietic stem and progenitor cells,19 preventing their exit into the afferent lymphatics and causing T-cell retention in secondary lymphoid tissues and proliferation of tissue-resident myeloid cells, respectively. Furthermore, elevation of local S1P can be involved in modulating the type of immune response by skewing the phenotype of lymphocytes, dendritic cells and macrophages.7 How S1P is elevated in tissues is still unclear; nonetheless, it is known that blood cells,20 and as we will discuss later, mast cells, are likely candidates for increasing S1P in the tissue environment since they can produce and release large quantities of S1P when stimulated. In contrast to the low levels of S1P in tissues, S1P concentrations in plasma and #   % ! #     \     ˜#$#   $ what is the “effective” concentration of S1P in circulation). Free S1P may be at least 40

126

MAST CELL BIOLOGY

times lower than the total S1P concentration because it is mostly bound to albumin or lipoproteins, but the bound form can be also physiologically active.21,22 The S1P-carrier proteins may provide a sink for excess S1P and/or a regulatory mechanism for protecting \       !  \    21,22 Recent data has uncovered # # ## !  \   $  {$     %   # processes: the maintenance of endothelial barrier integrity and lymphocyte recirculation between lymph nodes and periphery. Deletion of both SphK1 and SphK2 in an inducible mouse model renders these “S1P-less mice” with enhanced vascular permeability and induces lethality in response to mediators that induce vessel permeability.23 Resistance to lethality can be restored by transfusion of wild type erythrocytes,23 the cells responsible for maintaining high levels of S1P in blood.24,25 The proposed mechanism for the protective effect by S1P in the vascular barrier is its regulation of S1PR1 activity in endothelial cells.23 S1PR1 is normally abundant in these cells and S1P engagement of this receptor tightens endothelial adherens junctions26 protecting endothelium integrity. On the other hand, targeted deletion of SphK1 and 2 in the lymphatic endothelium, which results in ablation of the high concentrations of S1P in lymph but not in blood, causes impaired egress of lymphocytes from the lymph nodes.27 \ !$   #% #  of the tissue to blood/lymph S1P gradient by inhibition or genetic deletion of S1P lyase prevents the exit of lymphocytes from the lymph nodes,15,28 and that the presence of S1PR1 in lymphocytes is required for their egress from lymph nodes.29,30 Collectively, #   $ #  ‘    ! # !  # $  the lymph nodes is the S1PR1-dependent sensing by lymphocytes of an S1P gradient that exists between the lymph node (low levels of S1P) and the lymph (high levels of S1P).

&=$ =\kqkq* * €+ % & $ From the brief discussion above, it is apparent that production and degradation of S1P is tightly and dynamically regulated. This suggests that its export to the extracellular   !  # " $    $  {$  # $  cell surface expression of the multiple S1PRs must be critical in the shaping of a given physiological response. In this chapter we will summarize the recent knowledge on the regulation of S1P production and the function of S1PRs in mast cell biology and in the    & % <% #   # ##     = (Fc¡RI) induces the activation of SphK1 and SphK2 and the generation of S1P.8 We also know that the activities of SphKs are key to the activation and expression of S1PRs on mast cells, however, it should be recognized that SphKs are also essential to Fc¡RI-mediated mast cell activation independently of S1PR.8,31 When activated, mast cells secrete S1P into the extracellular space and produce gradients of S1P within resident tissues that   $  # {    $ 32-34 Moreover, dysregulation of S1P can affect the differentiation and responsiveness of mast cells and thus it may increase mast cell responses in health and (allergic) disease.31,35 S1P also seems to play a vital role in overcoming the severe (patho)physiological responses seen during anaphylaxis.9 Interestingly, its production in a nonhematopoietic compartment was found to be essential for the recovery from anaphylaxis. This indicates that the site of action for S1P may determine its role as a pro- or anti-allergic molecule.

S1P IN MAST CELL EFFECTOR FUNCTION AND ALLERGIC DISEASE

127

S1P IS GENERA R TED BY ACTIVATED MAST CELLS Fc¡*+ " \ It is well recognized that antigen-induced aggregation of IgE bound to Fc¡RI on mast cells elicits multiple biochemical events culminating in mast cell degranulation and the de novo synthesis and secretion of cytokines and lipid mediators.36 One of the biochemical events that occurs downstream of Fc¡RI engagement is the activation of # %#  ‹$     $ \ !   #%  ‚†ƒ^_`Œ cells, 37 *#  %        $$    $#  bone-marrow derived mast cells (BMMCs), liver-derived mast cells and human mast cells.31,33,38,39 Engagement of Fc¡RI elicits the partitioning of this receptor to lipid rafts where sphingolipids, such as SPH (the substrate for SphK), are enriched. The Src kinases Lyn and Fyn, which initiate early Fc¡RI signaling, are needed for the translocation of SphKs to lipid rafts and for their activation.33,40 SphKs interact with Lyn and Fyn, but not with Src or other tyrosine kinases such as Syk and the activation of SphK1 and 2 depends  #    #   <  # %   ^  caused a complete ablation of SphK K activation. 33,40 Further support for the importance of Fyn in SphK K activation was provided when Fyn expression was restored, which rescued the activation of SphK1 and SphK2.33 The adaptor protein Gab2 and the lipid < \Œ  %  # $   %   $      # activation of SphKs downstream of Fyn kinase.33    ƒ^    showed a delay in the activation of SphK K by IgE/Ag,40 yet these cells recovered to achieve an almost normal SphK K activation with time suggesting that Lyn may not be essential for the activity of SphKs but may play a role in facilitating their activation. It has been suggested that PLD 1 activity is also involved in both Fc¡RI-39 and FcaRI-mediated SphK1 activation41 in mast cells and human myeloid cells, respectively. This view is consistent %#  !$ 42 as well as our own (AO and JR R unpublished observation), that PLD activation is Fyn-dependent in mast cells. This suggests that PLD is also likely to  $  # !  # `%!  #   #   #% PLD may contribute to SphK K activation and the relationship to the translocation/activation of SphK1 and 2 in mast cells is not entirely clear. *  \    ;  ^ To directly address the importance of each of these isoforms in mast cell function, we  !       !         # #_ or both,31  # $    #  _    #43 Although our "  %# †

@   #  #_ ^  $  #%   that SphK2 was the major producer of S1P in these cells,31 mast cell degranulation was not consistently diminished in all tested cultures. This led us to believe that SphK1 and 2 might have some redundant functions. Unlike BMMCs derived from adult mice, mast   !   !     #_^     #% a reduction in the extent of the degranulation response and in the production of various cytokines and eicosanoids. In contrast, mast cells derived from liver progenitors of #^    #%      $ %#      # #  #_ # #     #_^   31 The robust and consistent defect seen in the degranulation response of mast cells derived from

128

MAST CELL BIOLOGY

the SphK2-null embryos clearly differed from the more mild and variable result seen in #_^$ †

@  !   $  &    # $  {       #    #    #^  adult mice (as discussed in the next paragraph), although another possibility is that the two mast cells populations are not phenotypically comparable. This latter possibility is not unexpected as the mast cell phenotype is known to vary depending on the environment they populate in vivo, or the growth factors used in vitro.44 Thus, certain populations of mast cells might differ in their use of SphK1 or SphK2 for compartmentalized or general S1P production. In agreement with the view that the microenvironment may be a determinant in how SphKs are used, we now have evidence (unpublished observation) that peritoneal     # #  #_^  $  #!    $  ˜#$#  # $ %   !   # #_^    mast cells). Thus, it is plausible that the relative importance of SphK1 and SphK2 depends  #     ˜* ™ *#    %# #   #  of SphK1 and SphK2 in human mast cells. In human bone marrow-derived mast cells only SphK1 mRN R A was found and silencing of this isoform by antisense oligonucleotide caused a reduction in degranulation.39 In CD34 -derived human mast cells 33 and in cord blood derived human mast cells (CBMC)38 both SphK1 and 2 were present and could be activated by Fc¡RI engagement. Silencing of SphK1 and SphK2 by RN R Ai in the latter cells and in the human mast cell line LAD238 showed that SphK1 was involved in degranulation, migration toward antigen and secretion of the chemokine CCL2/MCP1, whereas silencing of SphK2 reduced cytokine production (Table 1). +  )"  *  >    )  *%     Apart from the possible heterogeneity of the mast cells in those studies, there are other possible technical considerations when studying the relative involvement  # #   $  †

@  !   #  #_^  mouse models, which were generated independently of those we study, showed normal degranulation responses to Fc¡RI stimulation.45 Yet, the same report shows a reduction  #  $  #^  †

@ %#  # "  %  in vitro or in vivo by siRN R A (Table 1). Thus, it is important to consider the possible limitations of employing RN R Ai silencing or genetic deletion strategies in interpreting # $ *#    ! % # #^  †

@    genetic deletion versus RN R Ai silencing might be most easily ascribed to offf target effects of siRN R A or compensatory mechanisms from genetic deletion. The in vivo use of siRN R A   #     %$  "   $ $  cells, not just mast cells. Alternatively, the genetic deletion could cause changes in the environment that may impose epigenetic changes or compensatory mechanisms in bone  %    ˆ #   # #_^   $  $ $ # ! !  \   %#  # #^   # % ! of circulating S1P.43,46,47 As we demonstrated31 this had a marked effect on mast cell ! %#  %   #  #_^   showed normal histamine release. This was unexpected given that fetal-liver derived #_^    #% !  $ `%!  #   with the recent report that S1P can regulate the development of human mast cells derived from cord-blood progenitors into a mast cell type more akin with a skin-type mast cell.35

S1P IN MAST CELL EFFECTOR FUNCTION AND ALLERGIC DISEASE

129

Table 1. Reported role of SphK1 and SphK2 isoforms in mast cell responses in vitro and in vivo, using different experimental approaches SphK Isoform SphK1

T Type of SphK Alteration

T Type of MC

siRNA, Antisense oligo

†

@| ?Degranulation45 ? Ca response45 ? Cytokine45

Effect in Vitro

Effect on PCA (in vivo)

Effect on PSA (in vivo)

?45

?45

Normal ( (unpublished)

?Histamine31 ? Recovery9

`$ @| ?Degranulation38,41 ? Ca response41 ? Cytokine38 Knockout

BMMC

Normal31

(Strong response) ƒ @|

Normal31

\ @|

?Degranulation

(unpublished) SphK2

siRNA Knockout

BMMC

Normal45

Normal45

Hu MC

? Cytokine

BMMC

?Degranulation31

38

Normal45

LDMC

?Degranulation31 ? Ca response31 ? Cytokine31

PMC

?Degranulation

? (unpublished) Normal

histamine31 BRecovery9 (Poor response)

(unpublished) |†

@ $    %^ ! $$   – `$ @ #$ $$    (cord blood- or CD4 -derived); LDMC: mouse fetal liver derived mast cells; PMC: mouse peritoneal mast cells.

Thus, it is possible that environmental cues, such as high S1P levels in the blood and lymph,  $    #   # #     %  $ #  ! $$ # ˜\@™ #  #_^  mice results in a defective response (unpublished observation). Thus, this skin-localized challenge showed the expected impaired mast cell degranulation of the fetal liver-derived #_^    *#   %# % !  \  # <  #

     # # ## !   $ \  #    <   # #  ^  #  #_^      of normal response. Obviously, the function of the SphK K isoforms is more likely to be $$$ $  $^      $ 

130

MAST CELL BIOLOGY

*    # * 8; \  # $ ! $    !  #  $ pharmacological inhibitors, silencing RN R Ai approaches or genetic deletion models, all point to a critical role for SphKs in mast cell function and suggest that some potential overlap for the two isoforms might exist. What has not been studied in detail is the mechanism by which SphK1 or SphK2 affect mast cell function. Our studies on the  ! ^ !     #_^    # # $  cause for the overall impaired mast cell responsiveness was due to an impairment of $ {$"  \@ !31 In human mast cells, silencing RN R Ai approaches have implicated SphK1 in calcium release from intracellular stores.39 However, we uncovered no evidence of a role for SphKs or S1P in mobilizing calcium from endoplasmic $ ˜=‚™       # #  # < $ {$"  well known to be an essential process for Fc¡RI-induced mast cell degranulation and cytokine production48,49 and thus how SphK2 regulates calcium mobilization is of considerable interest. An essential characteristic of some calcium channels is their modulation by lipids50 and S1P has been shown to regulate calcium mobilization.6,51-53 #_^       $ \   $$ \` %## #     # $ {$"54-57 In contrast, S1P added exogenously to BMMCs had no effect on either Fc¡RI-induced calcium mobilization31 or store-operated calcium release-activated calcium current (iCRAC) in RBL-2H3,55 suggesting that the effect of S1P on calcium is likely to be intracellular and not through binding of its cell surface receptors on mast cells. As alluded to above, work in RBL-2H3 cells has shown that SPH is an inhibitor of store-operated calcium release-activated calcium current (iCRAC)55 and of the coupling of Orai1/CRACM1 to STIM1, the calcium sensor protein in the ER.58 Since SphK2 controls the intracellular levels of S1P and SPH in mast cells in the resting and activated states, it is possible that an elevation in S1P directly or indirectly leads to the opening of a calcium channel, or that a reduction of SPH releases the inhibitory effect on calcium channel function.31,55 In support of this view, Prieschl and colleagues32 proposed a rheostat model based on the observations that addition of exogenous SPH inhibited mast cell degranulation and leukotriene and TNF production, effects that could be effectively reversed by S1P. The model stipulates that the balance between SPH and S1P is decisive for the excitability of mast cells. Apart from the possibility that S1P or SPH may directly modify the function of an ion channel in mast cells, a more indirect mechanism is also possible. Recent studies in our lab suggest that reduced levels of nonselective cation channel Transient Receptor Potential Channel  ˜*‚\@™ ‚~     ^  †

@    $    # ! $ {$"   $  #  ˜‚  >‚ $$# results). Moreover, silencing of TRPC1 expression in WT BMMCs also resulted    $ {$"  !  $   !$  ^  †

@   ! #  ¡RI stimulation and we found that they also have lower baseline levels of S1P.33 Since S1P was recently shown to affect nuclear transcription by acting directly on histone deacetylases14 one possible scenario is that homeostatic S1P production may be involved in the regulation of certain genes critical for mast cell calcium responses, like TRPC1 and function. Thus, studies on S1P regulation of gene expression may shed further insights on the role of this lipid in regulating mast cell signals and function.

S1P IN MAST CELL EFFECTOR FUNCTION AND ALLERGIC DISEASE

131

Fc¡RI INDUCES S1P FORMATION AND EXPORT Fc¡RI-mediated activation of SphKs not only leads to the formation of S1P inside the cells,37,39 but also results in its export into the extracellular space.32,33,59 This suggests that the site(s) of action for S1P may be both intra- and extracellular. As previously mentioned, mast cells lacking SphK2, which do not produce intracellular S1P or export it, have a defect in calcium mobilization and degranulation and these defects are either not restored or only partially restored, respectively, when S1P is added exogenously. *# $ # #    \ ˜$#  #   $ {$"™   independent of its cell surface receptors on the mast cell.31 However, the function of \       % $   # # #    its binding to and regulating histone deacetylase activity,14 its intracellular targets are        #  #% # #    \     cell responses.59 In many cell types, the export of newly synthesized S1P may occur as a }   %#   \  $  #    { $   !  # $ { #$# # $  \     usually not readily detectable.6,60 Notably, mast cells and platelets can release copious amounts of S1P upon agonist stimulation8,20 suggesting that the S1P released plays a role beyond the autocrine modulation of cell function and may be an important paracrine component of mast cell and platelet regulation of immune responses.

#  $] Both platelets and mast cells secrete their granular contents by exocytosis. However, in mast cells, S1P is not pre-accumulated in granules and degranulation appears to be dissociated from S1P export.61,62  \   #  $ $ {^{ of S1P across the bilayer is not likely to occur and thus a facilitating transport mechanism must be in place. The ATP binding cassette (ABC) superfamily of transporters, which catalyze the transport of chemically diverse compounds across cellular membranes, have been involved in the transport of S1P, both from the outside to inside cells63 or from the inside to the outside of the cell.61,62,64 The ABCC7 ion channel CFTR R was implicated in the take up of S1P from the mileu.63 However, little is know about the role of this transporter in the uptake of S1P for mast cells. In contrast, several types of ABC transporters have    # {$"  \ $ †@†  * 64 an ABCA-like transporter in platelets,62 ABCC1 in the RBL-2H3 and human LAD2 mast cell lines,61 and ABCC1 and ABCG2 transporters in breast cancer cells.65 Inhibition of ABCC1 or ‚~    "    $ # ¡RI-stimulated export of S1P from mast cells, but it only partially reduced the constitutive export of S1P observed when SPH was added to the cells, suggesting other transporters or additional mechanisms of export might be present.61      !   \ ƒ^  mice, which have markedly elevated levels of S1P and enhanced constitutive as well as Fc¡‚^$ {$"  \  #  #%  $ $   †@ transporter (L. Wright, Y. Kitamura, AO and JR, unpublished observations), although its relevance to S1P export in these cells is still unknown. Recently, it was shown66 that in }  # #   _ %#  $$  #  #    ^Œ^##        "  \  $  # Spns2 gene resulted in the inability of yolk syncytial cells to release S1P during heart development, a critical event for the chemoattraction of myocardial precursors bearing the S1P receptor S1PR2 to

132

MAST CELL BIOLOGY

form the heart tube. A mutation in either Spns2 or in the S1PR2 genes led to the presence of a split heart; a phenotype that was rescued by injecting S1P, Spn2 mRNA or human Spns2 mRNA, but not the homolog Spns1 mRNA. Whether this type of transporter, or its homolog, may also function in mast cells has not been investigated. Mice carrying    †@      _   !  $‹$!   the type of transporter involved in the export of S1P and the contributory role of secreted \     {

FUNCTIONS OF S1P RECEPTORS IN MAST CELL RESPONSES Mast cells have been described to express two types of receptors for S1P, S1PR1 and S1PR2,59 #$#   !  $    #    ‚~  \’ as well, but not S1PR3 or S1PR5. Fc¡RI activation of SphKs and the production of S1P was shown to induce a ligand-dependent transactivation of S1PR1 and 2 enhancing mast cell functions.59 The transactivation of S1PR1 and S1PR2 can be readily recognized, as these receptors are endocytosed with `-arrestin, a common phenomena observed when a GPCR is activated.67 Antisense oligonucleotide-mediated silencing of S1PR1 or S1PR2 indicated that while S1PR1 was involved in the migration of mast cells toward low concentrations of antigen, S1PR2 participated in Fc¡RI-induced degranulation. Silencing of S1PR1 reduced and ectopic expression of S1P1 enhanced, respectively, chemotaxis of mast cells towards antigen without affecting degranulation. On the other hand, overexpression of S1P2 inhibited the chemotactic motility of RBL cells towards antigen.59 Moreover, S1PR2 mRN R A expression, but not S1PR1 mRN R A, was increased as a late consequence of Fc¡‚ $ *#   $    %#  \‚    # recruitment of mast cells to their site of action, while S1PR2 deters migration and contributes   $    # #        the conclusion that SphK K activation, S1P generation and export and S1PR1 engagement       #" ^    %##   ! SphKs, also have defective chemotaxis in response to antigen and SCF, but exogenous S1P in combination with Fc¡RI-generated signals can partly restore the motility defect.33 Similarly, pharmacological inhibition or genetic deletion of SphK K in mast cells resulted in reduced chemotactic motility towards antigen or SCF, but these cells were able to migrate normally towards S1P33,59 (AO and JR, unpublished results). Interestingly, inhibition of ABCC1-mediated S1P export blocked migration of mast cells to antigen in vitro and this was rescued by addition of S1P,61 suggesting that mast cell chemotaxis to antigen or SCF is, at least in part, dependent on mast cell derived S1P. The involvement of S1PR2 in degranulation is not as clear. Exogenous addition of S1P alone or in combination with Fc¡‚ $   $    $ in the RBL-2H3 cell line or in murine mast cells.33,59 However, RN R Ai silencing of S1PR2 or its genetic deletion in BMMCs resulted in a 40 to 50% reduction in Fc¡RI-induced degranulation. Of note, the degranulation response to ATP59 and to phorbol esters and ˜ˆ >‚$$# $™%   $\‚_^  †

@ suggesting a potential defect in the exocytotic machinery in these cells. Interestingly, inhibition of ABCC1-mediated S1P export did not affect Fc¡RI-induced degranulation, indicating that export of S1P by this transporter is needed for migration but not for degranulation.61 #$#    #   $         pool of S1P to the proximity of S1PR2, other possibilities include that the S1P/S1PR2

S1P IN MAST CELL EFFECTOR FUNCTION AND ALLERGIC DISEASE

133

axis functions independently of a transporter for the degranulation response, or perhaps in a model similar to that proposed for other S1PRs68 in which a constitutively active S1PR2 facilitates a signaling platform that promotes Fc¡RI signals. In this later model, # \‚_ $      \  # !   #$      Fc¡RI responses. For example, in normal tissues where the levels of S1P are low S1PR2 activity on mast cells could also be low. Thus, in tissues, mast cells might require higher amounts of antigen to trigger a response, whereas in mast cells that are in closer vicinity to the circulatory system (where the S1P concentration is high or under conditions where S1P is elevated) the activity of the S1PR2 would be higher, lowering the threshold of activation via the Fc¡RI. Nevertheless, this is hypothetical and more conclusive evidence for the role of this receptor in mast cells responses in vivo is needed. Our recent studies of systemic anaphylaxis in the S1PR2-null mice suggest that these mice have a normal immediate hypersensitivity response upon systemic antigen challenge. However, we uncovered a mast-cell independent contribution of this receptor in the recovery from #"  #$  ^      \‚_      directly assess its involvement in the mast cell responses during anaphylaxis or other allergic disorders. Paracrine Function of Mast Cell-Released S1P  $^       "   !   $ %#    can change given a particular circumstance. Antigen-stimulation of Fc¡RI, for instance, has been demonstrated to induce the production and release of S1P into the interstitium and #$ \         {   <% # # !  \     $  $ {  # 18 in the airways of asthmatic individuals hours after a challenge,69 and in the joints of rheumatic patients.70,71 The origin of the elevated S1P in tissues under these conditions is not known, but one reasonable candidate is the mast cell. Changes in S1P levels can have drastic effects in the surrounding tissue, not only by affecting angiogenesis and vascular permeability,1,72 but also by causing fundamental changes to the immune system.7,70,73 However, the     \  {     {   $  $  is likely that the particular challenge and the type of immune cells that respond to this #       \ $  {    # ! of S1P and/or increased expression of S1PRs can result in the accumulation of immune cells in the local site. Several mechanisms may account for this accumulation: induction of chemotaxis,29,59,74-78 retention of hematopoietic stem cell progenitors,19 and retention of mature T cells in the tissues; the latter two through increased LFA-1–ICAM-1 and VLA-4–VCAM-1-mediated adhesion to the afferent lymphatic endothelium.18 In vitro and in vivo experiments in murine systems suggest that S1P can also cause a shift in T-cell responses promoting TH2- and TH17-cell responses while disfavoring TH1-cell responses, thus potentially skewing responses toward allergic phenotypes. This, in part, can be mediated through the reported effects of S1P on dendritic cell maturation. S1P was shown to impair the ability of lipopolysaccharide (LPS)-stimulated human monocyte-derived DCs to cause naïve CD4 T-cells differentiation to T helper 1 (TH1)-cells and instead promoted TH2-cell differentiation.7,76 In this regard, it should also be kept in mind that S1P also seems to be important in the effector phase of allergic disease through control mast-cell responsiveness. Thus, in certain circumstances, it appears that S1P can play a ‘       {

134

MAST CELL BIOLOGY

 ^{    \ #      " \  $  %#    {    # ^{  _  # subtype,7 which explains the observed protective effect of S1P against atherosclerotic lesions.79 Furthermore, topical application in the skin80 or inhalation of S1P or the S1P mimetic FTY72081 suppressed, respectively, ear swelling in a model of allergic      % {   # # !   asthma model. The underlying mechanism for suppression appears to be the inhibition of dendritic or Langerhans cell migration to the lymph nodes and thus an impaired     ^  * 80,81 Furthermore, FTY720 also induced a decrease in the induction of TH1- and TH2-cell effector cytokines and the establishment of a stable synapse between DCs and T cells.81 Intriguingly, lesional skin biopsies of individuals with atopic dermatitis82 and psoriasis83 show increased expression of two enzymes involved in the degradation of S1P, S1P Lyase and S1P-phosphatase Type 2 (SPP2), respectively. The elevated presence of these enzymes could result in reduced levels of S1P and the  $  #  @ %# # !%  ^{    S1P was also suggested by the reduction in IL-1` and IL8 production by stimulated HUVEC or neutrophils when its degradation was blocked by RN R Ai silencing of SPP2.83 Alternatively, the SPP2-mediated conversion of S1P to SPH, which can be used to generate ceramide,17 or the breakdown of S1P by S1P-Lyase into metabolites that can be used for the synthesis of other lipids, particularly phosphatidylethanolamine,16 may also play a role in the course of these distinct diseases by affecting the lipid composition of the skin. Interestingly, application of an emulsion of ceramide to the skin appears to reduce the severity of atopic dermatitis.84-86 `%!   #     $  $#  the increase in expression of enzymes involved in the catabolism of S1P contributes to # !  #   $ #     ^{  \   #   #  $       ^{  \  producing other lipid products that improve the symptoms.

S1P-MEDIATED REGULATION OF THE MAST CELL PHENOTYPE AND RESPONSIVENESS Increased levels of S1P in the circulation were found to correlate with the ability to release granule-stored mast-cell allergic mediators (degranulation), such as histamine. This was unexpectedly uncovered when we compared the responses of mast cells derived   #^    !   #     $  =^  #" #^   #!   !   $ \  these changes in S1P levels are independent of the mast cells. 31 Genetic deletion of SphK1 resulted in the reduction of circulating S1P,31,46 because this isozyme is the major activity present in RBC, the cells most responsible for maintaining S1P levels in the blood. In contrast, genetic deletion of SphK2 caused an elevation in the circulating levels of S1P,31,47,87 perhaps due to a compensatory elevation of SphK1 activity in RBC. As previously mentioned, Fc¡‚^ $  #_^     !  $   !  $  `%!  %# #_^  mice were challenged systemically, in an IgE/Ag-dependent manner, the mast cell response (as measured by histamine release) was similar to WT mice suggesting that the higher !  \  #  $ $ #_^     $   In contrast, S#^   %#   #%    $  ! 

S1P IN MAST CELL EFFECTOR FUNCTION AND ALLERGIC DISEASE

135

had decreased histamine release upon a systemic challenge. This apparent disconnect between the in vitro and in vivo results appeared to indicate that the exposure to high or low levels of S1P caused an alteration of the mast-cell phenotype31 (Fig. 2 and Table 1). As discussed earlier, it was also possible that the phenotype of the mast cells differentiated  !    { #  !! #       # # response. However, additional experiments (AO and JR, unpublished observations) showed #   ˜$$™ =^#  #^ #_^   #%  normal or defective mast cell response, respectively, that mirrored the results observed

Figure 2. Dual role of sphingosine-1-phosphate in the regulation of systemic anaphylaxis. While S1P can enhance the signaling and function of mast cells (left panels), which initiate the allergic response, it alleviates the symptoms of the anaphylactic shock acting in a mast cell-independent manner (right panel). Exposure of mast cell progenitors to higher levels of S1P in the cellular environment can change the differentiated phenotype and/or enhance the responsiveness of mast cells. The effect of S1P on mast cells can be indirect, involving monocytes/macrophages, or direct (left, upper panel). Thus, under circumstances where S1P is increased, mast cells can become more sensitive to a stimulus and thus {$ #           $ \ ! # !  # (left lower panel). The activation of SphK2 by Fc¡RI is necessary for the activation of calcium channels at the plasma membrane and for the degranulation of mast cells. The S1P generated is exported to the outside of the cell, where its concentration increases locally and can thus engage the S1PRs on the mast    # #    # {   *#   #  mast cells triggers SphK1 activity and the production of S1P from nonmast cell sources (right panel). This S1P activates S1PR2 receptors in the vasculature, regulating the vascular tone and thus blood  $  %##  $     #  $     #    # $ in the recovery from anaphylaxis. SphK1 and S1P production can also activate S1PR1 receptors, which prevent vascular leakage and this may also contribute to recovery.

136

MAST CELL BIOLOGY

in vitro (Table 1). This was not due to a difference in the number of mast cells in the skin or differences in granule content since the local (cutaneous) response to compound 48/80, a secretagogue, was comparable in all the mutant and WT mice. This suggests #   #" #  # !   $ \  {$ # responsiveness of the mast cell and thus the sensitivity and/or severity of this response. In the skin, where levels of S1P are inherently low, the role of mast-cell extrinsic S1P    !     {$ Additional data in models other than SphK-null mice also support the idea that in vivo regulation of S1P homeostasis (extrinsic to the mast cell) can regulate mast cell responsiveness. Mice from an 129Sv genetic background, which undergo strong TH2-cell responses and anaphylaxis,88 have higher levels of circulating S1P as compared to C57BL/6 mice, which have modest TH2-cell responses.89 Increasing the levels of circulating S1P in C57BL/6 mice, by using an inhibitor of S1P lyase,15 results in mice with enhanced mast cell responses as determined by increased histamine in the plasma following a systemic challenge. The level of the response resembles that observed in 129Sv mice, suggesting a link between S1P levels and the enhanced mast cell phenotype ˜ˆ  >‚ $$#  !™ ‚    # "$   human cord blood progenitors to both S1P and stem cell factor enhances the rate of differentiation of these progenitors to mast cells and causes them to become more like a connective tissue type mast cell (containing chymase and tryptase in their granules) rather than a mucosal type (containing tryptase) mast cells, which is the normal when cells are cultured in the presence of stem cell factor alone.35 The mechanism for how S1P causes the developmental shift in the phenotype of human mast cells appears to be partially indirect, involving the production of IL-6 by cells of monocyte/macrophage lineage.35 Our unpublished observations on the effect of S1P during murine BMMCs differentiation indicate that chronic treatment with S1P induces genetic changes that give rise to a hyper-responsive mast cell. This may be a direct action of S1P on the mast cells, but whether this is mediated by a S1PR R on the mast cell remains to be determined. Thus, S1P homeostasis in circulation or the tissue environment, which can be dysregulated by genetic causes or by dietary or environmental factors, may result in alterations in the phenotype of mast cells and the susceptibility to particular allergic reactions. Although the discussed data supports an effect of the extrinsic S1P in vivo as a $    ! #   $ # % $  < S1P, by conditional deletion of both SphK1 and SphK2, had normal mast cell responses to an anaphylactic challenge when challenged with strong stimuli,23 suggesting normal mast cell function in the absence of S1P. Additional studies are needed to determine if the susceptibility of mast cells to S1P in the environment or within the mast cell depends on the type or the strength of the stimulus.

RECOVERY FROM ANAPHYLAXIS IS POSITIVELY REGULATED BY S1P FROM A NON-MAST CELL SOURCE S1P receptors are ubiquitously expressed and affect multiple physiological functions in various tissues.7 Thus, it is likely that, beyond their role in mast cell biology and function, #    {$ #   #      \‚ S1PR2 and S1PR3, are known to regulate the vascular system affecting vascular tone, vascular permeability and heart rate.90-92 In endothelial cells, S1PR1 preserves vascular

S1P IN MAST CELL EFFECTOR FUNCTION AND ALLERGIC DISEASE

137

integrity and regulates vascular permeability.23,91,93 S1PR3 functions in myocardial contractility and in the regulation of bradycardia and hypertension induced by S1P;94 and S1PR2 increases vascular permeability and regulates renal, mesenteric and local blood {%  ! $  91,95,96 All these processes are vastly altered during an allergic reaction like systemic anaphylaxis. Thus, it is not surprising that S1P produced during #"     #  $ {$ # !   #   acting on the vascular system. Recently, we discovered9 that S1PR2 plays an important role in counteracting the vasodilator actions of histamine and therefore the hypotension associated with systemic anaphylaxis (Fig. 2). We found that histamine drives S1P production by activating SphK1 and this is required for the engagement of S1PR2 and the recovery from anaphylaxis. Genetic deletion of SphK1 or S1PR2 disrupts this regulatory loop during histamine or IgE/Ag-induced anaphylaxis, causing a severe hypotension, with a consequent delay in the renal rate of histamine excretion and thus a delay in the recovery from anaphylaxis.9 Importantly, the delayed recovery from anaphylaxis could be $  #^    !$ ‘  \ $  #" `%!  \ ‘ #    #  !   \‚_^    that the presence of this receptor is required for S1P-induced rescue from anaphylaxis. Other studies suggest that S1PR1 may also play a role in recovery from anaphylaxis.23 Inducible genetic deletion of both SphK1 and SphK2 results in mice that lack circulating or tissue S1P. When challenged with IgE/Ag or PAF, these mice undergo a severe anaphylactic response with resulting lethality. The IgE (SPE-7 clone) and PAF used in this study are potent inducers of mast cell degranulation. A massive    $  {$  !$ " ! %  !  # %  $ to the loss of S1PR1 function, since lethality was partially prevented by injection of a S1PR1 agonist.23  # # %   %# #^  mice were challenged with PAF and these mice also showed enhanced vascular {$ " !23    %    !    !$ {$ " ! % &*  #^    $   = ˜$ a H1-DNP¡ ™  #^$ #" #$# #^   clearly showed more severe anaphylaxis. Thus, whether S1PR1 or S1PR2 is dominant in recovery from anaphylaxis may well depend on the stimuli. Alternatively, models %#  #  # $  $   { #   # response to a stimulus, since recovery is common.97 Regardless, these two different studies suggest that generation of S1P during anaphylaxis and/or the presence of S1P in the circulation have a protective effect via the activation of either or both S1PR1 and S1PR2. While S1PR1 plays a role in the maintenance of the vascular barrier integrity, S1PR2 regulates the vascular contractility that allows for the recovery from hypotension resulting from a systemic anaphylactic challenge. It is known that, in humans, the two main anaphylactic triggers are IgE- or IgG-mediated and the type and the prevalence of released mediators differs, with PAF release being more abundant in IgG-mediated anaphylaxis. Thus, from a therapeutic perspective, a combination of S1PR1 or S1PR2 agonists used at different ratios may prove useful in the treatment of anaphylaxis. In this regard, we found that treatment of WT mice with S1P during histamine-induced anaphylaxis expedited their recovery from anaphylaxis at a similar     %#   #  #     #" `%!  administration of adrenaline carries some risks, particularly in the elderly population, including cardiac arrhythmias, myocardial infarction and hypertensive intracerebral bleeds.98,99 Since the S1PR2 receptor does not mediate bradycardia, tachycardia or

138

MAST CELL BIOLOGY

{$   $  !!       $   {%  appear to be mostly vascular,95          $$ alternative for anaphylaxis treatment. As a note of caution, it is important to point out that S1PR2 has the opposite role of S1PR1 in vascular permeability, as its activation in the endothelial cells can lead to increased vascular permeability. However, the fact that # \‚    "   #   # \‚_^  in our model of histamine-induced anaphylaxis did not reveal differences in vascular permeability, provides an argument for further exploration of S1PR2 agonists in counteracting anaphylaxis. Overall, another important conclusion suggested from the studies on anaphylaxis is that SphK/S1P K is a “Janus” regulator of the systemic allergic response (Fig. 2). On one hand, it is part of the signals generated upon engagement of Fc¡RI and functions to enhance the activation of mast cells. Also, the presence of S1P in the microenvironment may render mast cells with a more responsive phenotype. On the other hand, SphK/S1P K also seems to regulate other aspects of the allergic response that are independent of the mast cell.9 In the case of systemic anaphylaxis, although it enhances mast cell immediate responses, it also mediates the recovery from shock, thus affecting positively and negatively, respectively, the onset and duration of anaphylaxis. Such dual roles for physiological mediators are not unusual in biology since, in a similar way, a given cytokine may have distinct roles as an initiator or attenuator of the immune responses.100-102

CONCLUSION SphK1, SphK2, their product S1P and the receptors for S1P are all emerging as important regulators of allergic responses. Their roles extend beyond the regulation of the effector function of mast cells and other immune cells to the regulation of the surrounding environment and the (patho)physiological response. Due to the complexity of their actions and the various types of cells involved, further investigations must be aimed    #       # !    # $ to the resulting outcomes. The models that completely eliminate the expression of these molecules (through genetic deletion) in the entire mouse have been useful so far, but they are limited in allowing a further understanding of the role of S1P in different cell types and compensatory mechanisms may complicate the interpretation of their observed !!  " $  # !   #   #^    # # #  !!  # !     {103 However, studies where pharmacological inhibitors of SphK K or RN R Ai for SphK1 were   #% # #  !    $ { %  reduced.71,104 Although the reasons for these discrepancies are not entirely clear, one of the possibilities is that the pharmacological inhibitors or the silencing RN R Ai may target a population(s) of cells that is more susceptible to these treatments and are essential in #     *$^     # #  \‚       %$  ##    !$ #  *# $ be particularly valuable when considering SphKs or S1PR2 as targets for therapy and in furthering our understanding of their role in health and disease. Nevertheless, we conclude from the work cited herein that SphKs, S1P and S1PRs are key components in the regulation of allergic responses and beyond.

S1P IN MAST CELL EFFECTOR FUNCTION AND ALLERGIC DISEASE

139

ACKNOWLEDGEMENT The research of the authors included herein was supported by the intramural research program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health.

REFERENCES 1. Saba JD, Hla T. Point-counterpoint of sphingosine 1-phosphate metabolism. Circ Res 2004; 94:724-734. 2. Schwab SR, Cyster JG. Finding a way out: Lymphocyte egress from lymphoid organs. Nature Immunol 2007; 8:1295-1301. 3. Allende ML, Proia RL. Sphingosine-1-phosphate receptors and the development of the vascular system. Biochim Biophys Acta 2002; 1582:222-227. 4. Kono M, Allende ML, Proia RL. Sphingosine-1-phosphate regulation of mammalian development. Biochim Biophys Acta 2008; 1781:435-441. 5. Hannun YA, Obeid LM. Principles of bioactive lipid signalling: Lessons from sphingolipids. Nat Rev Mol Cell Biol 2008; 9:139-150. 6. Spiegel S, Milstien S. Sphingosine-1-phosphate: An enigmatic signalling lipid. Nat Rev Mol Cell Biol 2003; 4:397-407. 7. Rivera J, Proia RL, Olivera A. The alliance of sphingosine-1-phosphate and its receptors in immunity. Nat Rev Immunol 2008; 8:753-763. 8. Olivera A, Rivera J. Sphingolipids and the balancing of immune cell function: Lessons from the mast cell. J Immunol 2005; 174:1153-1158. 9. Olivera A, Eisner C, Kitamura Y et al. Sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 are vital to recovery from anaphylactic shock. J Clin Invest 2010; 120:1429-1440. 10. Taha TA, Hannun YA, Obeid LM. Sphingosine kinase: Biochemical and cellular regulation and role in disease. J Biochem Mol Biol 2006; 39:113-131. 11. Olivera A, Spiegel S. Sphingosine kinase: A mediator of vital cellular functions. Prostaglandins 2001; 64:123-134. 12. Sanchez T, Hla T. Structural and functional characteristics of S1P receptors. J Cell Biochem 2004; 92:913-922. 13. Rosen H, Goetzl EJ. Sphingosine 1-phosphate and its receptors: An autocrine and paracrine network. Nat Rev Immunol 2005; 5:560-570. 14. Hait NC, Allegood J, Maceyka M et al. Regulation of histone acetylation in the nucleus by sphingosine-1-phosphate. Science 2009; 325:1254-1257. 15. Schwab SR, Pereira JP, Matloubian M et al. Lymphocyte sequestration through s1p lyase inhibition and disruption of S1P gradients. Science 2005; 309:1735-1739. “ †<   ƒ ƒ †   \     $  #  !  > † @# 10.1074/jbc.M109.081489. 17. Hannun YA, Luberto C, Argraves KM. Enzymes of sphingolipid metabolism: From modular to integrative signaling. Biochemistry 2001; 40:4893-4903. 18. Ledgerwood LG, Lal G, Zhang N et al. The sphingosine 1-phosphate receptor 1 causes tissue retention by inhibiting the entry of peripheral tissue T-lymphocytes into afferent lymphatics. Nat Immunol 2008; 9:42-53. 19. Massberg S, Schaerli P, Knezevic K -Maramica I et al. Immunosurveillance by hematopoietic progenitor cells   < # $#  #   #  $ @ _••”– Œ€€’^••— 20. Yang L, Yatomi Y, Miura Y et al. Metabolism and functional effects of sphingolipids in blood cells. Br J Haematol 1999; 107:282-293. 21. Yatomi Y. Plasma sphingosine 1-phosphate metabolism and analysis. Biochim Biophys Acta 2008; 1780:606-611. 22. Sattler K, Levkau B. Sphingosine-1-phosphate as a mediator of high-density lipoprotein effects in cardiovascular protection. Cardiovasc Res 2009; 82:201-211. 23. Camerer E, Regard JB, Cornelissen I et al. Sphingosine-1-phosphate in the plasma compartment regulates   {^$ !$ <   > @ ! _••€– €—”^—”€ 24. Pappu R, Schwab SR, Cornelissen I et al. Promotion of lymphocyte egress into blood and lymph by distinct sources of sphingosine-1-phosphate. Science 2007; 316:295-298.

140

MAST CELL BIOLOGY

25. Hanel P, Andreani P, Graler MH. Erythrocytes store and release sphingosine 1-phosphate in blood. FASEB J 2007; 21:1202-1209. 26. Lee MJ, Thangada S, Claffey K KP et al. Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate. Cell 1999; 99:301-312. 27. Pham TH, Baluk P, Xu Y et al. Lymphatic endothelial cell sphingosine kinase activity is required for lymphocyte egress and lymphatic patterning. J Exp Med 2010; 207:17-27, S11-14. 28. Vogel P, Donoviel MS, Read R et al. Incomplete inhibition of sphingosine 1-phosphate lyase modulates immune system function yet prevents early lethality and nonlymphoid lesions. PLoS One 2009; 4:e4112. 29. Matloubian M, Lo CG, Cinamon G et al. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 2004; 427:355-360. 30. Cyster JG. Specifying the patterns of immune cell migration. Novartis Found Symp 2007; 281:54-61; discussion 61-54, 208-209. 31. Olivera A, Mizugishi K, Tikhonova A et al. The sphingosine kinase-sphingosine-1-phosphate axis is a determinant of mast cell function and anaphylaxis. Immunity 2007; 26:287-297. 32. Prieschl EE, Csonga R, Novotny V et al. The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc¡RI triggering. J Exp Med 1999; 190:1-8. 33. Olivera A, Urtz N, Mizugishi K et al. IgE-dependent activation of sphingosine kinases 1 and 2 and secretion of sphingosine 1-phosphate requires Fyn kinase and contributes to mast cell responses. J Biol Chem 2006; 281:2515-2525. 34. Jolly PS, Rosenfeldt HM, Milstien S et al. The roles of sphingosine-1-phosphate in asthma. Mol Immunol 2002; 38:1239-1245. 35. Price MM, Kapitonov D, Allegood J et al. Sphingosine-1-phosphate induces development of functionally mature chymase-expressing human mast cells from hematopoietic progenitors. FASEB J 2009; 23:3506-3515. Œ“ ‚!  >     $ $    ! >   @ $ _••“– 117:1214-1225. 37. Choi OH, Kim JH, Kinet JP. Calcium mobilization via sphingosine kinase in signalling by the Fc¡RI antigen receptor. Nature 1996; 380:634-636. 38. Oskeritzian CA, Alvarez SE, Hait NC et al. Distinct roles of sphingosine kinases 1 and 2 in human mast cell functions. Blood 2008; 111:4193-4200. 39. Melendez AJ, Khaw K AK. Dichotomy of Ca2 signals triggered by different phospholipid pathways in antigen stimulation of human mast cells. J Biol Chem 2002; 277:17255-17262. ’• ' } ~ ˆ!   † ^@  =   =  !  # <      $ to Fc¡RI are mediated by its interaction with lyn kinase. Mol Cell Biol 2004; 24:8765-8777. 41. Melendez A, Floto RA, Gillooly DJ et al. FcaRI coupling to phospholipase D initiates sphingosine <^ $ }  !$   < > † @# €€—– _”Œ€Œ€Œ^€’•_ 42. Choi WS, Hiragun T, Lee JH et al. Activation of RBL-2H3 mast cells is dependent on tyrosine phosphorylation of phospholipase D2 by Fyn and Fgr. Mol Cell Biol 2004; 24:6980-6992. 43. Mizugishi K, Yamashita T, Olivera A et al. Essential role for sphingosine kinases in neural and vascular development. Mol Cell Biol 2005; 25:11113-11121. 44. Galli SJ, Kalesnikoff J, Grimbaldeston MA et al. Mast cells as “Tunable” effector and immunoregulatory cells: Recent advances. Annu Rev Immunol 2005; 23:749-786. 45. Pushparaj PN, Manikandan J, Tay HK K et al. Sphingosine kinase 1 is pivotal for Fc epsilon RI-mediated mast cell signaling and functional responses in vitro and in vivo. J Immunol 2009; 183:221-227. ’“  ƒ < * % `       # <      # by FTY720. J Biol Chem 2004; 279:52487-52492. 47. Zemann B, Kinzel B, Muller M et al. Sphingosine kinase type 2 is essential for lymphopenia induced by the immunomodulatory drug FTY720. Blood 2006; 107:1454-1458. 48. Ma HT, Beaven MA. Regulation of Ca2 signaling with particular focus on mast cells. Crit Rev Immunol 2009; 29:155-186. 49. Vig M, Kinet JP. The long and arduous road to crac. Cell Calcium 2007; 42:157-162. 50. Hardie RC. Trp channels and lipids: From drosophila to mammalian physiology. J Physiol 2007; 578:9-24. 51. Xu SZ, Muraki K, Zeng F et al. A sphingosine-1-phosphate-activated calcium channel controlling vascular smooth muscle cell motility. Circ Res 2006; 98:1381-1389. _ <  `$ @> # ^##  $ $ {$"    ^  calcium entry. J Biol Chem 2003; 278:27540-27547. Œ † #% @>  > < ‚@   @$ {$"     $   $ sphingosine 1-phosphate accumulation. J Biol Chem 2001; 276:11712-11718. 54. Titievsky A, Titievskaya I, Pasternack M et al. Sphingosine inhibits voltage-operated calcium channels in GH4C1 cells. J Biol Chem 1998; 273:242-247.

S1P IN MAST CELL EFFECTOR FUNCTION AND ALLERGIC DISEASE

141

55. Mathes C, Fleig A, Penner R. Calcium release-activated calcium current (icrac) is a direct target for sphingosine. J Biol Chem 1998; 273:25020-25030. 56. Budde K, R LS, Nashan B et al. Pharmacodynamics of single doses of the novel immunosuppressant FTY720 in stable renal transplant patients. Am J Transplant 2003; 3:846-854. 57. Blom T, Bergelin N, Slotte JP et al. Sphingosine kinase regulates voltage operated calcium channels in GH4C1 rat pituitary cells. Cell Signal 2006; 18:1366-1375. 58. Calloway N, Vig M, Kinet JP et al. Molecular clustering of Stim1 with Orai1/CRACM1 at the plasma membrane depends dynamically on depletion of Ca2 stores and on electrostatic interactions. Mol Biol Cell 2009; 20:389-399. 59. Jolly PS, Bektas M, Olivera A et al. Transactivation of sphingosine-1-phosphate receptors by Fc¡RI triggering is required for normal mast cell degranulation and chemotaxis. J Exp Med 2004; 199:959-970. 60. Hobson JP, Rosenfeldt HM, Barak LS et al. Role of the sphingosine-1-phosphate receptor Edg-1 in PDGF-induced cell motility. Science 2001; 291:1800-1803. 61. Mitra P, Oskeritzian CA, Payne SG et al. Role of ABCC1 in export of sphingosine-1-phosphate from mast cells. Proc Natl Acad Sci USA 2006; 103:16394-16399. 62. Kobayashi N, Nishi T, Hirata T et al. Sphingosine 1-phosphate is released from the cytosol of rat platelets in a carrier-mediated manner. J Lipid Res 2006; 47:614-621. “Œ †$‘$ ƒ@ † #%^& @  @   @      $ $ $< of sphingoid base phosphates and lysophosphatidic acid: Modulation of cellular activity of sphingosine 1-phosphate. J Biol Chem 2001; 276:35258-35264. 64. Honig SM, Fu S, Mao X et al. FTY720 stimulates multidrug transporter- and cysteinyl leukotriene-dependent T-cell chemotaxis to lymph nodes. J Clin Invest 2003; 111:627-637. 65. Takabe K, Kim RH, Allegood JC et al. Estradiol induces export of sphingosine-1-phosphate from breast cancer cells via ABCC1 and ABCG2. J Biol Chem. 2010; 285(14):10477-86. ““ %#   ~# * ` š   *# #    _ $     }  # myocardial precursors. Science 2009; 323:524-527. 67. Kovacs JJ, Hara MR, Davenport CL et al. Arrestin development: Emerging roles for beta-arrestins in developmental signaling pathways. Dev Cell 2009; 17:443-458. 68. Waters CM, Long J, Gorshkova I et al. Cell migration activated by platelet-derived growth factor receptor is blocked by an inverse agonist of the sphingosine 1-phosphate receptor-1. FASEB J 2006; 20:509-511. 69. Ammit AJ, Hastie AT, Edsall LC et al. Sphingosine 1-phosphate modulates human airway smooth muscle  $ #   {   %   # =† > _••– __^_’ 70. Kitano M, Hla T, Sekiguchi M et al. Sphingosine 1-phosphate/sphingosine 1-phosphate receptor 1 signaling  #$ !$ ‚$  !     {   "   #  Rheum 2006; 54:742-753. R ” ƒ &Ÿ  % & # ``   ^{    # < $  {   #  > $ _••—– ——••^—•” 72. Lee OH, Kim YM, Lee YM et al. Sphingosine 1-phosphate induces angiogenesis: Its angiogenic action and signaling mechanism in human umbilical vein endothelial cells. Biochem Biophys Res Commun 1999; 264:743-750. 73. Oskeritzian CA, Milstien S, Spiegel S. Sphingosine-1-phosphate in allergic responses, asthma and anaphylaxis. Pharmacol Ther 2007; 115:390-399. ”’ &} * @# ƒ @#" >   ~$  <    <  !! ‹$    # 1-phosphate receptor. Nat Immunol 2007; 8:1337-1344. 75. Roviezzo F, Del Galdo F, Abbate G et al. Human eosinophil chemotaxis and selective in vivo recruitment by sphingosine 1-phosphate. Proc Natl Acad Sci USA 2004; 101:11170-11175. 76. Idzko M, Panther E, Corinti S et al. Sphingosine 1-phosphate induces chemotaxis of immature and modulates cytokine-release in mature human dendritic cells for emergence of Th2 immune responses. FASEB J 2002; 16:625-627. ””  ` } => ƒ##  #  ^$    {  immunity. Biochim Biophys Acta 2002; 1582:168-174. 78. Czeloth N, Schippers A, Wagner N et al. Sphingosine-1 phosphate signaling regulates positioning of dendritic cells within the spleen. J Immunol 2007; 179:5855-5863. 79. Nofer JR, Bot M, Brodde M et al. FTY720, a synthetic sphingosine 1 phosphate analogue, inhibits development  #    %^    ^   @ $ _••”– •^•— 80. Reines I, Kietzmann M, Mischke R et al. Topical application of sphingosine-1-phosphate and FTY720 attenuate allergic contact dermatitis reaction through inhibition of dendritic cell migration. J Invest Dermatol 2009; 129:1954-1962. 81. Idzko M, Hammad H, van Nimwegen M et al. Local application of FTY720 to the lung abrogates experimental asthma by altering dendritic cell function. J Clin Invest 2006; 116:2935-2944.

142

MAST CELL BIOLOGY

—_  =š \ < * ƒ       #         ^ \@‚ J Invest Dermatol 2006; 126:1187-1189. 83. Mechtcheriakova D, Wlachos A, Sobanov J et al. Sphingosine 1-phosphate phosphatase 2 is induced during {   @  _••”– €”’—^”“• 84. K Kucharekova M, Schalkwijk J, Van De Kerkhof PC et al. Effect of a lipid-rich emollient containing ceramide 3 in experimentally induced skin barrier dysfunction. Contact Dermatitis 2002; 46:331-338. 85. Kang JS, Yoon WK, Youm JK K et al. Inhibition of atopic dermatitis-like skin lesions by topical application of a novel ceramide derivative, K6PC-9P, in Nc/Nga mice. Exp Dermatol 2008; 17:958-964. 86. Chamlin SL, Kao J, Frieden IJ et al. Ceramide-dominant barrier repair lipids alleviate childhood atopic dermatitis: Changes in barrier function provide a sensitive indicator of disease activity. J Am Acad Dermatol 2002; 47:198-208. 87. Mizugishi K, Li C, Olivera A et al. Maternal disturbance in activated sphingolipid metabolism causes pregnancy loss in mice. J Clin Invest 2007; 117:2993-3006. —— š# š @#  ~ $ $ š   @$   !  {$   ‚^$ mast cell activation and allergic responses. J Immunol 2007; 179:740-743. 89. Rivera J, Tessarollo L. Genetic background and the dilema of translating mouse studies to humans. Immunity 2008; 28:1-4. 90. Igarashi J, Michel T. Sphingosine-1-phosphate and modulation of vascular tone. Cardiovasc Res 2009; 82:212-220. 91. Lee JF, Gordon S, Estrada R et al. Balance of S1P1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature. Am J Physiol Heart Circ Physiol 2009; 296:H33-42. 92. Means CK, Brown JH. Sphingosine-1-phosphate receptor signalling in the heart. Cardiovasc Res 2009; 82:193-200. 93. Singleton PA, Dudek SM, Chiang ET et al. Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1 and alpha-actinin. FASEB J 2005; 19:1646-1656. 94. Forrest M, Sun SY, Hajdu R et al. Immune cell regulation and cardiovascular effects of sphingosine 1-phosphate receptor agonists in rodents are mediated via distinct receptor subtypes. J Pharmacol Exp Ther 2004; 309:758-768. 95. Lorenz JN, J Arend LJ, Robitz R et al. Vascular dysfunction in S1P2 sphingosine 1-phosphate receptor knockout mice. Am J Physiol Regul Integr Comp Physiol 2007; 292:R440-446. 96. Sanchez T, Skoura A, Wu MT et al. Induction of vascular permeability by the sphingosine-1-phosphate receptor-2 (S1P2R) and its downstream effectors rock and pten. Arterioscler Thromb Vasc Biol 2007; 27:1312-1318. 97. Lieberman P, Camargo CA, Jr., Bohlke K et al. Epidemiology of anaphylaxis: Findings of the american college of allergy, asthma and immunology epidemiology of anaphylaxis working group. Ann Allergy Asthma Immunol 2006; 97:596-602. 98. El-Shanawany T, Williams PE, Jolles S. Clinical immunology review series: An approach to the patient with anaphylaxis. Clin Exp Immunol 2008; 153:1-9. 99. Pumphrey RS. Lessons for management of anaphylaxis from a study of fatal reactions. Clin Exp Allergy 2000; 30:1144-1150. 100. Yoshida H, Nakaya M, Miyazaki Y. Interleukin 27: A double-edged sword for offense and defense. J Leukoc Biol 2009; 86:1295-1303. 101. Mosser DM, Zhang X. Interleukin-10: New perspectives on an old cytokine. Immunol Rev 2008; 226:205-218. •_ ˆ   \ ž ^{  < @# _•••– ”“_^”_ •Œ #$ > #  \  ‚ƒ   ~  $  #  {    # kinase 1 knockout mice. FEBS Lett 2006; 580:4607-4612. 104. Lai WQ, Irwan AW, Goh HH et al. Distinct roles of sphingosine kinase 1 and 2 in murine collagen-induced arthritis. J Immunol 2009; 183:2097-2103.

CHAPTER R9 DOWN-REGULATION OF MAST CELL RESPONSES THROUGH ITIM CONTAINING INHIBITORY RECEPTORS ƒ      ƒ!^# | Department of Pharmacology and Experimental Therapeutics, Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel |     >*3<< '=

Abstract:

*# $   #   # $   !     system against invading pathogens and micro-organisms. In general, immune cells are activated for disparate functions, such as proliferation, production and release of mediators and chemotaxis, as a result of interactions between ligands and their matching immunoreceptors. This in turn leads to the recruitment and activation of a cascade of second messengers, via their regulators/adaptors, that determine the net effect of the initial response. However, activation of cells of the immune  $  # $    $   % ! and inhibition to avoid excessive or inappropriate responsiveness and to maintain homeostasis. Loss of inhibitory signals may disrupt this balance, leading to various pathological processes such as allergic and auto-immune diseases. In this chapter, we will discuss down-regulating mechanisms of mast cells focusing on immunoreceptor tyrosine-based inhibition motifs (ITIM)-containing inhibitory receptors (IR).

INTRODUCTION Mast cells are the main effector cells of allergic reactions as well as are important regulators of a number of pathophysiological processes. Mast cells can be activated by the binding of ligands to various receptors expressed on the cells. Nevertheless, the main mechanism of mast cell activation in allergy is through the binding of an    # ¯‚^= " \!  !  %##     through immunoreceptor tyrosine-based activation motifs (ITAM)-bearing receptors,

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 143

144

MAST CELL BIOLOGY

are counterbalanced by negative or inhibition signals transmitted by immunoreceptor tyrosine-based inhibition motifs (ITIM). This is especially important to maintain the balance between activation and inhibition and to avoid hyperresponsiveness. In this chapter we will discuss in depth the down-regulation of IgE-mediated mast cell responses through ITIM-mediated inhibition signals.

CELL ACTIVATION Cells of the immune system can be activated through a wide array of receptors, such as receptors for the Fc portion of different immunoglobulin subclasses (FcR), cytokines receptors, T-cell antigen receptor (TCR), the B-cell antigen receptor (BCR), Toll-like receptors (TLR), integrin receptors and G protein coupled receptors including those for   #<     =2 and adenosine. Ligands for the aforementioned receptors mediate their effects on cells through the activation of receptor-linked signal transduction pathways. A pivotal mechanism of cell activation is carried out by Fc receptors. The extracellular   #     #    #   #    involved. On the other hand, the intracellular domains of the Fc receptors are crucial for transducing signals into the cells and eventually for determining the duration and intensity of the initiated response.1 The cytoplasmic portion of theFc receptors contains immunoreceptor tyrosine-based activation motifs (ITAMs) through which the immune system transduces positive signals. U Upon ligand recognition and receptor co-aggregation, tyrosine residues in the ITAMs are phosphorylated by Src family protein tyrosine kinase(s) (PTK(s)). The phosphorylated ITAMs serve as docking sites for the SH2 domains of Syk family PTKs, such as ZAP-70 or Syk. Syk family PTKs phosphorylate a series of substrates, leading to the formation of membrane-proximal scaffolds. In turn, important effector molecules, such as phospholipase C (PLC), are recruited leading to calcium signaling, as well as Ras activation, resulting in stimulation of the ERK K pathway and cellular activation.2 In this chapter we will focus primarily on the IgE-mediated mast cell activation # *# #    ##^  =  ˜¡RI) on mast cells consists of three subunits: _, ` and a. The _ subunit is unique to this receptor while the a subunit is common to other receptors such as FcaRIIa. The ` chain is important for stabilizing the receptor and for amplifying its signaling. Upon cross-linking of IgE bound to the Fc¡RI receptor with the multivalent antigen, Fc¡RI complexes aggregate and lead to a transphosphorylation of the ITAMs situated in the cytoplasmic regions of the ` and a chains. Tyrosine residues in the ITAMs are then phosphorylated by the src family kinase Lyn to initiate the signaling cascade13 as above.

$ +%=++&+(%k&$* +%&+(% (^ +

)%$ *$(%$   $       # #    #       $  #  {   `%!   # #  ! # {   $      ! a chronic process. It is for this reason that multiple mechanisms exist to terminate or to %^ $ # !  #  $  {    # # {   !  # $    #  $ 

SHPs SHP-2, SHP-1

MHC-1 molecules Collagens CD38, _v`3

Lmirr1 -

Gp49B1

PIR-B

LAIR-1

PECAM-1

CD200R -

-

CD300a SIRP- _

˜ƒƒ‚†’™|

ILT/ T CD85

LAIR-1

PECAM-1

CD200R MAFA

CD72

CD 100

CD200 E-cadherin

SHP-1,2

_v`3

SHP-1 49

SHIP SHIP, SHP-2

SHP-1 (SHP-2)

SHIP; SHPs SHP-1,2

Unknown CD47

SHP-1

-

SHIP

Phosphatase Recruited

Siglec-8

Immune complexes lgG Abs 6vsulfo-sLex

Ligand

Fc aRIIB

Murine Counterpart

Fc aRIIB

Human

|‚ %  $   "   $  #$    #   !

C-type lectin like

Ig-superfamily

Subfamily

2 ITIMs

NPXY motif 1 ITIM

2 ITIMs

2 ITIMs

4 ITIMs

4 ITIMs

1 ITIM; 1 ITIM-like 4 ITIMs 2 ITIMs

1 ITIM

Inhibitory Component

Table 1. Human and murine inhibitory receptors expressed on mast cells

Eosinophils Myeloid, hematopoetic neurons Neutrophils, Macrophages, NK B cells, DC, Monocytes, Macrophages Most cells of the immune system Endothelial, Neutrophils, NK, Monocytes, Platelets Basophils NK, Basophils, U937 (human) NK, CD8-T cells (mouse) B-cell, basophils

Eosinophils

B cells

Additional Cells Where Receptor Is Expressed

DOWN-REGULATION OF MAST CELL RESPONSES THROUGH ITIM 145

146

MAST CELL BIOLOGY

secretion of arachidonic acid metabolites (i.e., prostaglandins, thromboxanes, leukotrienes)    ^{  < $#  ƒ^— ƒ^  *~^_. Moreover, other      $ $  { $ #     elastase from neutrophils and perforin from NK K cells. Therefore, one way to end the {      #  $     $#   Indeed, a natural mechanism of self “extinguishing” the acute phase response occurs due to the relatively short halff lives of the lipid metabolites.3 Similarly, production of ^{  < ˜ ƒ^• * ^`) and down-regulation and desensitization  $       $  #    {       #    {4,5 This can occur through two main signaling pathways: an intrinsic one mediated by the mitochondria; and an extrinsic one mediated by death receptors. Both pathways rely on a family of intracellular cysteine proteases called caspases.6 The expression and function of two death receptors, FAS/CD95R R and TRAIL-R, has been shown in both murine and human mast cells.7   #    {     # $# # $ or catabasis process. This is an active process that leads to the reduction or removal  $<      {  *# $      $ #  $ #%   # #  ^{  pro-resolution lipid mediators such as lipoxins, resolvins and protectins.8 In addition to the mechanisms mentioned above regulating immune responses, there has been a growing interest in a family of receptors that mediate inhibitory responses and are therefore named “inhibitory receptors”. As opposed to the positive signals transmitted through ITAM-bearing receptors, negative regulation of the immune system is controlled by immunoreceptor tyrosine-based inhibition motifs (ITIM).

ACTIVATION AND DOWN-REGULATION OF MAST CELL RESPONSES: GENERA R L Mast cells are highly granulated, Fc¡RI-bearing tissue-dwelling cells that develop       "  @Œ’ @” ˜™  @Œ $ # {$ of stem cell factor (SCF). Mast cells have a “lead” role in allergic reactions, but are also !!  $    $      $$ diseases and in host-defense against bacterial and parasitic infections9 (also reviewed in other chapters). By their activation and consequent release of mediators, mast cells cause the early symptoms of allergy and orchestrate and actively participate in the later #     #    ^  {      $  %#    !   #       # $ Mast cells can be activated by various stimuli in addition to “the classical” binding of an allergen to the Fc¡RI-IgE complex. For example, SCFwhose receptor Kit is expressed on mast cells can induce cell differentiation, maturation, chemotaxis and survival; and also potentiate antigen-mediated mast cell activation.10 Other examples of mast cell activation are through the binding of a ligand to other receptors such as: TLRs; and cytokine, chemokine, complement and neuropeptide receptors, all of which are also expressed on mast cells.11,12 Moreover, basic or polybasic substances such as some endogenous mediators (i.e., substance P, bradykinin, neurotensin, eosinophil derived major basic protein, etc.) or drugs (i.e., codeine, morphine, desferroxamine), or synthetic peptides (compound 48/80) can similarly stimulate mast cells.10

DOWN-REGULATION OF MAST CELL RESPONSES THROUGH ITIM

147

+%=++&(* * *$$&(*J +&+  +&  % +&

#    %      #  }  ~  %#  their ligands are primarily MHC-I molecules. The interaction of MHC-I with inhibitory receptors on NK K cells leads to the abrogation of signaling pathways and therefore prevents the cell’s cytotoxic activity against self.13 Inhibitory receptors have now been shown to be expressed on multiple cell types in the immune system, including T cells, B cells, mast cells, eosinophils and neutrophils (Table 1). The inhibitory receptors belong either to the Ig-receptor super-family or to the c-type (calcium dependent) lectin super-family. As mentioned above, inhibitory receptors      * ‹$ @ *        $   six amino acid sequence, I/V/LxYxxL/V (x denoting any amino acid), present in the intracytoplasmic domain of transmembrane molecules with inhibitory properties which, when phosphorylated, recruit SH2 domain-containing cytosolic phosphatases.14,15 The Ig-like super-family inhibitory receptors are characterized by the presence of a single V-type Ig like domain in the extracellular portion. This family includes FcaRIIB, sialic acid binding Ig-like lectins (siglecs), signal regulatory protein _ (SIRP_), Ig-like transcripts/leukocyte immunoglobulin receptors (ILTs/LIRs), killer cell Ig-like receptors (KIR), platelet endothelial cell adhesion molecule (PECAM-1), carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM-1), leukocyte-associated Ig-like receptors-1 (LAIR-1), CMRF-35H and paired Ig-like Type 2 receptor _ (PILR-A). Inhibitory receptors that belong to the C-type lectin superfamily derive their name from the homology in their extracellular domain to the C-type lectins. These are known to be $^  %##   #  # $#      carbohydrate-recognition domain (CRD) (Table 1).16,17  #%  $       #     one or several ITIMs, suppresses cell activation by promoting dephosphorylation reactions. Upon ligation/activation of ITIM-containing receptors, tyrosine residues within these motifs become phosphorylated after the activation of receptor tyrosine kinases or Src family member tyrosine kinases (SFKs). This leads to the recruitment of the protein phosphatases, Src homology 2 domain-containing tyrosine phosphatase (SHP)-1 and SHP-2, or the lipid phosphatase, Src homology 2 domain-containing inositol 5- phosphatase (SHIP) 1. SHP-1/2 dephosphorylate tyrosine-containing signaling molecules and therefore counterbalance the action of tyrosine kinases. On the other hand, SHIP1 dephosphorylates phosphatidylinositol 3,4,5 trisphosphate at the 5v position, thereby terminating PI3K-mediated signaling pathways.12 The dephosphorylation of these signaling proteins leads to the dampening of cellular activation. It is important to note that the different ITIM-bearing inhibitory receptors mediate their actions through different effector phosphatases. This partially determines the net effect of the inhibitory response. Moreover, although inhibitory receptors are usually characterized by an ITIM in their cytoplasmic tails, some of the newly discovered inhibitory receptors contain either an ITIM-like sequence or other tyrosine-containing motifs that are crucial for the inhibitory functions of the receptor. Apart from ITIMs, certain immunoreceptors contain immunoreceptor tyrosine-based switch motifs (ITSMs). Like ITIMs, ITSMs can associate with SHP-1/2 or with SHIP. ITSMs can also bind adaptor molecules such as SH2-domain-containing protein 1A (SH2D1A) and EWS-activated transcript 2 (EAT-2). Moreover, ITSMs can bind to Src family kinases and the p85 regulatory subunit of PI3K.18 Therefore, ITSMs can

Figure 1. Schematic representation of ITIM-dependent inhibition of Fc¡RI-mediated activation. Mast cell Fc¡RI aggregation by receptor-bound IgE with an antigen leads to the phosphorylation of the receptors immunoreceptor tyrosine-based activation motifs (ITAMs). Tyrosine residues in the ITAMs are then phosphorylated by the kinase Lyn. Another tyrosine kinase (Syk) is also recruited to the complex and phosphorylates LAT. The latter activates PLCa to degrade PI(4,5)P2 to IP3 and DAG. IP3 induces intracellular Ca2 } %   " $ {$" ƒ*   !   <    $   a downstream activation of MAPKs (JNK, p38, ERK). Subsequently, mast cell degranulation, cytokine and lipid mediator production occurs. Co-aggregation of an inhibitory receptor with Fc¡RI leads to the phosphorylation of the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the inhibitory receptor, leading to the recruitment of tyrosine phosphatases i.e., SHP-1/2 and SHIP. SHP-1/2 dephosphorylates the Fc¡RI ` and a chains as well as Syk. SHIP dephosphorylates PIP3 to for PIP2 thus abrogating downstream signaling and Ca2 {$" *#     !    $  %  <  $  abrogated. (The scheme was built using the “pathway builder” at www.proteinlounge.com).

148 MAST CELL BIOLOGY

DOWN-REGULATION OF MAST CELL RESPONSES THROUGH ITIM

149

transduce an activating or inhibitory signal depending on the immune receptor that they are associated with and the cell type in which they are expressed. The inhibitory receptor Lmir-3, which is expressed on murine mast cells and other myeloid cells, has been shown     <  $ ƒ ^Œ  !   $ %  which (Y241, Y289) are in the ITIM sequence, one in the ITSM and two (Y276, Y303) in the binding motifs for p85 or Grb2, respectively. The existence of the latter indicates a possible activating potential, in addition to its inhibitory one. *# _†’       ~     $ !  inhibitory signals. The inhibitory signals are mediated by the phosphorylation of its third ITSM and subsequent recruitment of SHP-1/2 and SHIP.19 2B4 has also been shown to be expressed (and functional) on murine mast cells.    # #  * #  #  #% # $   conditions, ITAMs can also transduce inhibitory signals. This led to the term inhibitory ITAMs or ITAMi. One of the known examples of such dual effects is mediated through immunoglobulin IgA and its receptor, Fc_RI. Upon multimeric ligand binding and Fc_RI    {        %    monomeric or low-valency ligand, with no sustained receptor aggregation, an inhibitory ^{    $ *#     # #     with the ligand determines the balance between inhibitory and activating functions of Fc_RI. Moreover, ITAM-containing adaptors, FcR Ra and DNAX-activating protein 12 (DAP12), have been shown to carry out dual functions. FcR Ra has been shown to be involved in the inhibition of IgG-mediated phagocytosis in monocytes and IgE-mediated degranulation in mast cell transfectants.20 Additionally, DAP12, associated with various immunoreceptors, mediated the inhibition of macrophages responses to pathogen-initiated signals through TLRs.21 Studies have demonstrated inhibition of activating responses of heterologous receptors such as FcaR or Fc¡RI, using anti-Fc_RI Fab antibodies. These inhibitory effects are associated with the recruitment of SHP-1 as observed in ITIM-mediated inhibition, as well as a resultant decrease in Fc¡RI-induced ERK, LAT and Syk phosphorylation.20 In summary, although ITAM-activating responses are counter regulated by ITIM-regulated inhibition, ITAMi-mediated inhibition has emerged as a new aspect of negative regulation to cell activation. However, ITAMi inhibition appears to act as a more general mechanism in the long term maintenance of balance in the immune system. It is important to note that inhibitory receptors exist in human as well as murine cells, while some of the human receptors do not always have a murine counterpart.

MAST CELL-ASSOCIATED INHIBITORY RECEPTORS & +@> #; FcaRIIB The Fca‚† % #  #    %## # * $ % recognized, having been shown to inhibit B-cell receptor (BCR)-mediated activation in vitro.22 It is a receptor for the immunoglobulin G constant (Fc) region and a Type-I transmembrane protein consisting of 291 amino acids containing one ITIM which has been shown to be expressed in both murine bone marrow derived mast cells (BMMC)

150

MAST CELL BIOLOGY

and human cord blood derived mast cells (CBMC). FcaRIIB displays different inhibitory effects on MC proliferation and activation. In murine BMMC, co-aggregation of FcaRIIB with Fc¡RI results in the inhibition of IgE-mediated mast cell degranulation. The intact ITIM in the cytoplasmic tail of FcaRIIB is crucial to this effect.23,24 Similarly, co-aggregation of FcaRIIB with Fc¡RI inhibits degranulation and cytokine secretion. This response is mediated by phosphorylation of tyrosine residues on FcaRIIB and subsequent recruitment of SHIP-1 but nor SHP-1/2.14,24-26 FcaRIIB can also negatively regulate mast cell proliferation when co-aggregated with Kit27 through the activity of SHIP-1. Siglecs Members of the siglec family have one variable (V-type) region located at the N-terminal domain which binds sialic acid residues and between 1-16 C2-type Ig-like domains in the extracellular portion.22 The majority of the members of this family contain ITIM or ITIM-like domains. These receptors are expressed on various cell types of the innate and adaptive immune systems. However, the siglecs that are expressed on mast cells belong mainly to the CD33-related siglecs.28 Human mast cells express siglec-2, siglec-3, siglec-5, siglec-6, siglec-8 and siglec-10.29 Notably, a relatively high level of siglec-5, 6 and 8 proteins, all of which contain one ITIM motif, have been detected on   $    {30-32 Among the members of this family that are expressed on human mast cells, siglec-8 is the one that has been most studied. This receptor contains one ITIM and one ITIM-like domain that upon antibody-induced coligation of the receptor, undergo activation, recruit `\^    %  #  ! #$#  %    on eosinophils, where self aggregation by a mAb led to apopotosis, ligated siglec-8    %       # ¡RI-triggered histamine and prostaglandin D2 release. Interestingly, it had no effect on the release of other synthesized mediators such as cytokines.33,34 As with many of the inhibitory receptors expressed on human cells, murine counterparts also exist in the siglec family. However, the murine functional homolog of the human siglec-8, siglec-F, is not expressed on murine mast cells but is expressed on murine eosinophils. Although the natural ligand for siglec-8 has long been unknown, recent studies have proposed mucins as the potential ligand, based on their ability to engage with siglecs and to subsequently induce apopotosis on monocytes.35,36 Moreover, in a more recent paper, it has been shown that eosinophils can undergo apoptosis when incubated not only with anti-siglec-8 Abs, but also with a $ #   “±^$^ƒx glycan.37 CD300a CD300a (IRp60 R or CRMF-35) is expressed on NK K cells, MCs, T-cell subsets, granulocytes, monocytes and dendritic cells.15,38-40 It is one of seven members in a family of stimulatory as well as inhibitory Type I transmembrane glycoproteins that control and modulate leukocyte responses. CD300a contains four ITIMs in its cytoplasmic tail, three of them being “classical” (LHYAN A L, VEYSTV, LHYASV) and the fourth “non classical” (SDYSVI).15 *# $  @Œ•• %     ~  where its co-aggregation resulted in down-regulation of the cells’ cytolytic activity. Subsequently, CD300a’s expression and function was further characterized in human mast cells. In CBMC, co-aggregation of CD300a with IgE-bound Fc¡RI, led to inhibition

DOWN-REGULATION OF MAST CELL RESPONSES THROUGH ITIM

151

of IgE-induced (but not of compound 48/80-induced) `-hexosaminidase, tryptase and IL-4 release. Concomitantly, an increase in CD300a phosphorylation, recruitment of SHP-1 and SHIP-1, decrease in Ca2 {$"     < ##  %  detected. Moreover, CD300a cross-linking inhibited SCF-mediated CBMC survival.38   % ^   _-IgE/_-CD300a and _-Kit/_-CD300a, produced to crosslink the activating with the inhibitory receptors, abrogated Kit-mediated CBMC differentiation, survival and activation; and IgE dependent activation. In the context of mast cell-related disorders, when added to the malignant human mast cell line HMC-1, %#    $! ! # @Œ••    #  release without affecting cells survival. In CBMCs, the inhibitory effect of CD300a on Kit was found to be mediated by a rapid phosphorylation of CD300a, recruitment of SHIP-1 but not of SHP-1 and by subsequent dephosphorylation of Syk and LAT. CD300a and other members of this family have mouse orthologs, named CLM or CMRF-like molecules. The murine homologue of CD300a, LMIR-1 (or CLM-8), shares almost 80% homology with the human receptor and is expressed and functional on murine BMMC. In these cells, CD300a/Lmir-1 is capable of recruiting SHP-1, SHP-2 and SHIP.41,42 CD300a has been shown to have a crucial role in the down-regulation or inhibition of allergic responses. This was demonstrated in murine models of allergic peritonitis, passive cutaneous anaphylaxis and acute asthma, where use of the murine ^   _-CD300a(Lmir-1)/_-IgE proved to be effective in abrogating these allergic reactions.43 The ligand for CD300a remains unknown. However, evolutionary data reveals that CD300a is one of the human genes that shows strong positive selection 44,45 hinting that its potential ligand might have similarly gone through a strong positive selection as well.46 SIRP-_ SIRP-_ belongs to the Ig-superfamily and is characterized by three Ig-like domains in the extracellular portion. The intracellular domain contains four tyrosine residues which form two ITIMs that recruit SHP-2 and SHP-1, thus negatively regulating signal transduction pathways.47 SIRP-_ has been shown to be expressed on human basophils and CBMC as well as on HMC-1 cells. A murine homologue of SIRP-_ exists, but there is no evidence regarding its expression on mast cells. Functionally, SIRP-_ inhibited mast cell degranulation when coligated with Fc¡RI and decreased the phosphorylation of Fc¡RI ITAMs. This was attributed to the recruitment of the tyrosine phosphatases SHP-1/2.22 Moreover, it reduced intracellular Ca2 } {$"  " $ Ca2 and the activation of the MAP kinases Erk1 and Erk2. This resulted in inhibition of IgE-induced mast cell mediator release. The ligand SIRP-_ #     @’”  integrin-associated transmembrane protein which is expressed on many cell populations. The interaction of the ligand with its receptor inhibits not only mast cell related responses but also FcaR dependent/independent phagocytosis by macrophages. Moreover, SIRP-_ has been shown to inhibit production of IFN-a by mature dendritic cells, suggesting that it inhibits the development of Th2 cytokines that drive allergic responses. LILRs LILRs belong to a family of receptors that are expressed on a wide range of cells such as B cells, dendritic cells, monocytes and NK K cells. In humans, these receptors are

152

MAST CELL BIOLOGY

   #   $   #  ^<    #$  and organization: Ig-like transcripts (ILTs/CD85), killer-cell Ig-like receptors (KIRs) and leukocyte-associated Ig-like receptors (LAIRs (see below). It has been recently shown that LILRs are developmentally regulated in mast cells, suggesting a role in their maturation and differentiation. Human cord blood-derived progenitor mast cells (hPrMCs) express the inhibitory LILRB2, LILRB3 and LILRA2 on their surface. However, despite the presence of mRN R A for multiple LILRs in mature CBMCs, LILR R protein expression on the surface of these cells was not detected.48    {%     mature human mast cells showed high intracellular expression of LILRB5 while none of the other inhibitory LILRs were detected. Gp49B1 (mouse LILRB4), is a member of the Ig superfamily expressed constitutively on the surface of mast cells, neutrophils, macrophages49 and natural killer (NK) cells. Gp49B1 is similar to FcaRIIB in that it contains two C2-type, Ig-like domains. However, gp49B1 has two cytoplasmic ITIMs.50 The ITIM motifs in gp49B1 down-regulate NK K cell and T-cell activation signals that lead to cytotoxic activity. In mouse BMMCs, gp49B1 binds SHP-1 and SHP-2 recruited from the cytoplasm upon tyrosine-phosphorylation. Gp49B1-mediated inhibition of Fc¡‚^ ! !  $  `\^^  BMMC.51  ’€† $! # ! {   IgE-dependent mast cell activation in vivo.52 It has also been shown that injection of LPS into gp49B1 null mice leads to hemorrhage, thrombosis and tissue neutrophilia. These $  # ’€† $  ƒ\^$ { #   ! critical innate protection against a pathologic response to a bacterial component.53 The   ’€† #     #   _v`3, based on in vitro cell-cell and cell-protein binding studies. The interaction of _v`3 with Gp49B1 on BMMC has been shown to inhibit antigen-induced IgE-mediated cell activation.54 Paired Ig-Like Receptor B (PIR-B) \‚^†   #          #   #  of the immunoglobulin-like receptors. PIR-B is expressed by many types of hemopoietic cells, including B lymphocytes, dendritic cells, monocyte/macrophages, granulocytes, megakaryocytes/platelets and mast cells.55 This inhibitory receptor, as well as its activated counterpart, is produced by murine mast cells. PIR-B was found to be preferentially expressed on the cell surface, where it is constitutively tyrosine phosphorylated and associated with iSHP-1. After coligation with Fc¡RI, PIR-B inhibited IgE -mediated mast cell activation and release of serotonin.56 The human counterparts of PIR-A and PIR-B are considered to be the activating and inhibitory types of leukocyte Ig-like receptors/ CD85. Out of four ITIMs that the PIR-B receptor contains, two of them were found to be able to recruit SHP-1 and possibly SHP-2 when tyrosine phosphorylated and this leads to inhibition of cell activation.57 The ligand for PIR-B appears to be various mouse major histocompatibility complex class I (H-2) molecules. Indeed, stimulation of PIR-B with H-2 tetramer on B cells, leads to intracellular phosphotyrosine signaling.58 LAIR-1 LAIR-1 (CD305) is a human Type I transmembrane glycoprotein which contains a single extracellular C2-type Ig-like domain and two ITIMs in its cytoplasmic tail. #$#  #      $$        ƒ‚^

DOWN-REGULATION OF MAST CELL RESPONSES THROUGH ITIM

153

is expressed on almost all cells of the immune system. In the RBL-2H3 mast cell line, cross linking of LAIR-1 was able to inhibit Fc¡RI-mediated degranulation. Both ITIMs of this receptor are required for the full inhibition of degranulation in these cells, although the receptor is still partially active with one functional ITIM.59 The murine homologue of LAIR-1 (mLAIR-1) shares 40% identity with the human receptor. '<  #    %#  #!    #   LAIR-1 is not a cell-bound molecule. Collagens which are now known as the ligands  ƒ‚^    % <   " $  " ˜=@ ™    $ regulation.60 Human LAIR-1 recruits SHP-1 and SHP-261,62 but not SHIP.63 However, mLAIR-1, recruits SHP-2 but not SHP-1. The inhibitory function of LAIR-1 is not completely phosphatase-dependent but can also be mediated through Csk, a negative effector that can inactivate Src family kinases, in cells where the phosphatases activity is abrogated or limited.63 CD200 Receptor (CD200R) CD200R R is a Type-I membrane glycoprotein, containing two Ig-like domains and which is expressed on MC64 and basophils.65 Unlike the majority of immune inhibitory receptors bearing ITIMs, CD200R R does not contain a classical ITIM motif but instead contains three tyrosine residues in its cytoplasmic tail which may be critical for function. Previous studies have shown the expression of CD200R R on human CBMC and skin mast cells. CD200R R has also been detected on murine bone marrow derived mast cells (BMMC) and murine skin mast cells. In human mast cells, CD200R R produced an inhibition in degranulation that was enhanced by cross linking the anti-hCD200R Abs. This inhibition did not require coligation to an activating receptor such as Fc¡RI, although this did further enhance inhibition. This differs from most inhibitory receptors whose inhibitory effect depends on co-aggregation/ coligation to the balancing activating receptor. In murine mast cells, engagement of CD200R by its soluble ligand (CD200) did not lead to the inhibition of mast cell degranulation or cytokine production. However, overexpression of the receptor on these cells made them sensitive to anti-mCD200R Abs mediated inhibition. This demonstrates that CD200R activity depends on its density across the cells surface. Furthermore, the inhibitory effect of CD200R R was also evident in vivo in a model of passive cutaneous anaphylaxis (PCA). ‘  ^@_••‚    ˜!™      !   decreased PCA skin reactions in a dose dependent manner. As mentioned, CD200R R does not contain an ITIM sequence in its cytoplasmic domain. However, it has been shown that upon receptor engagement, CD200R R is rapidly phosphorylated and recruits inhibitory adaptor proteins such as Dok1 and Dok2.66 Two of the tyrosine residues of CD200R R (Y286, Y297) have been found to be critical for the receptor- mediated inhibitory effect. One of these tyrosines is located in a conserved phosphotyrosine binding site (NPXY297) for the signaling protein Shc. This motif mediates phosphorylation of Dok1 and Dok2 which subsequently bind RasGAP and SHIP, leading to a downstream inhibition of the RasMAPK K pathways in the cell activation. @_••    "   #$  $      # cells, dermal nerve bundles, hair follicles and subsets of glandular epithelial cells, but not on keratinocytes, Langerhans cells or the majority of mast cells and macrophages of the dermis in both murine and human skin sections.67 Nevertheless, mast cells were

154

MAST CELL BIOLOGY

detected in close proximity to CD200-expressing cells in skin tissues of human skin and %#      "   # $  < (PECAM-1) PECAM-1, also known as CD31, is a newly discovered member of the Ig superfamily containing six extracellular Ig domains and two ITIMs.68,69 PECAM-1 was found to be expressed mostly at the lateral junctions of endothelial cells and at lower levels on neutrophils, monocytes, platelets, NK K cells, T/B-cell subsets and on mast cells both of human and murine origin. This receptor has been shown to play a role in a number of biological processes, including leukocyte transmigration, cell migration, angiogenesis, cell adhesion, as well as modulation of intracellular signaling.70 In RBL-2H3 cells, following Fc¡RI clustering alone (without any need for receptor co-aggregation), PECAM-1 was shown to undergo rapid tyrosine phosphorylation on its ITIMs.71 Based on plasmon resonance studies, it has been shown that PECAM’s *   `\^_ %# ##    `\^ %#  %  72 The ligands for PECAM-1 include the adhesion molecule _v`373 (which is also the ligand for gp49B1 previously described) and CD38, a cell surface molecule involved in the regulation of lymphocyte adhesion to endothelial cells.74 & @&;  #; Mast Cell Function-Associated Antigen (MAFA)

* ^˜* ™  %##%   in RB R L-2H3 cells in which most of its inhibitory effects have been studied. Mouse and human homologues of the rat MAFA were thereafter discovered. Mouse MAFA was renamed as killer cell lectin-like receptor subfamily G member 1 (KLRG1), which exhibited an 89% similarity at the amino acid level to rat MAFA. As opposed to the rat receptor, KLRG1 was found to be expressed only by lymphokine- activated NK K cells and by virus-activated CD8 T cells. It was not expressed on mice mast cells. Moreover, a human MAFA-like receptor #     ^ƒ #   ’œ    #    *# #$ receptor, differing from the rat MAFA, is expressed not only on mast cells and basophils, but also on various types of NK K cells and the monocyte-like cell-line U937. Human MAFA has been proposed to likely regulate responses to receptors other than the Fc¡RI.75 In RBL-2H3 cells, MAFA has been shown to suppress the Fc¡RI secretory response and cytokine synthesis.76       $   #  cell secretory responses prior to Fc¡RI activation. However, co-aggregation of MAFA with Fc¡‚ #   #%      # #    degranulation.77 Additionally, MAFA clustering has been found to negatively regulate the    ‚†ƒ^_`Œ  *# % !        # $   cells arrested in the sub-G phase.75 Using surface plasma resonance analysis it has been shown that the cytoplasmic tail of MAFA, containing an ITIM sequence, binds SHIP and SHP-2 but not SHP-1.78 However, it has been shown that SHIP is the key phosphatase mediating the receptor’s inhibitory effect. This involvement of SHIP is thought to mediate the recruitment of a multimolecular complex (Shc–SHIP–Dok–RasGAP) to the plasma membrane, where, RasGAP down-regulates the Ras-induced Raff 1/MEK/E K RK K signaling pathway by decreasing RasGTP levels. This in turn may eventually lead to a decrease in

DOWN-REGULATION OF MAST CELL RESPONSES THROUGH ITIM

155

gene transcription and in synthesis of cytokines regulated by Erk-1/2. On the other hand, according to various studies, MAFA does not interfere with the Fc¡RI-induced Fyn– Gab2–PI3K K signaling pathway, which is essential for PKB and the Jnk pathway activation. Regarding the ligand for MAFA, it was originally thought that the receptor binds MHC-I molecules. This hypothesis was suggested since there is a great homology between MAFA’s extracellular domain and the CRD of other C-type lectins. However, there was no demonstrable interaction between murine MAFA and MHC-I or MHC-II molecules. It has also been implied that MAFA binds to saccharides following investigation of its binding capacity. Members of the classical cadherin family were shown to act as ligands for murine KLRG179-81 and the ligation of the receptor by E-cadherin led to an inhibition of CTLs induction as well as the lytic activity of an NK K cell line in vitro. Recently, it has been demonstrated that the ligand for human MAFA is also the human E-cadherin.82 CD72 CD72, also termed Lyb-2, is an ITIM-containing, 45 kDa Type II transmembrane protein83 predominantly expressed on B-lineage cells in both mouse and human.84 The precise function of CD72 remains unclear. However, studies have shown that anti-mouse-CD72  $     #   ##  $     B cells.85 Moreover, it induces an increase in MHC class II expression on B cells86,87 and mobilization of small amounts of cytoplasmic free Ca2 in those cells. The natural ligand  #  #     @••  #  ’ ˜’™88 Recently, CD72 has been found to be expressed on human mast cell lines (LAD2, HMC1.1, HMC1.2) as well as on CD34 peripheral blood-derived mast cells. CD72 contains two ITIMs thus including this receptor in the inhibitory receptor family. Upon tyrosine phosphorylation, one of the receptor’s ITIM binds SHP-1 and the other binds Grb2. On mast cells, coligation of CD72 with Kit by an anti-CD72 Ab (BU40) or the recombinant human CD100 (rCD100) and SCF respectively, led to CD72 phosphorylation and the subsequent recruitment of SHP-1. This resulted in dephosphorylation of SFKs and ERKs that are crucial in Kit-mediated human mast cell responses. CD72 ligation suppressed HMC1.2 growth and led to the reduction of Kit-dependent growth of human mast cells, SCF-induced chemotaxis, MCP-1(CCL2) production and SCF-enhancement of IgE-dependent degranulation. In contrast, CD72 ligation with Fc¡RI alone was unable to inhibit mast cell degranulation.89

INHIBITORY RECEPTORS AS THERA R PEUTIC TOOLS?

    $#     %  ^  {   ˜# # ‘$! ™        # &  world’s population with an increasing incidence each year. Some of these diseases might in certain cases lead to morbidity. Even with the proper use of current treatments such as glucocorticoids and several symptomatic drugs, symptoms and severe side effects may continue to occur and no ideal drug is yet available. Therefore, there remains a critical   %  #     ^       $$    way. The expression of inhibitory receptors on mast cells makes them attractive targets for new drug designs for allergy and other mast cell-driven diseases. Indeed, some of the studies on inhibitory receptors aimed for therapeutic development are at an advanced stage.

156

MAST CELL BIOLOGY

&# # !   #  #    a‚† ^   recognizing both the receptor and Fc¡RI were generated. When tested in vitro, these antibodies showed a suppressive effect on basophil as well as on human mast cell activation.90

 !     # ^   # $   $   #a-hFc¡ showed an inhibitory effect in both in vitro and in vivo studies using a murine passive cutaneous anaphylaxis model (PCA), as well as in skin test responses in R Rhesus macaques allergic to dust mite.91,92 For CD200R, in a murine model of PCA, treating the mice with      @_••‚   #      ^ decrease in cellular response.67 Another member of the inhibitory receptors superfamily, @Œ•• #    !    #       †^  antibodies targeting both mast cells and eosinophils have been generated and proven to be effective in abrogating allergic responses. Indeed, as described, in both in vitro and in !! $ # ^   _-IgE/_-CD300a and _-CCR3/_-CD300a abrogated allergic responses mediated by these cells.39,42,43

CONCLUSION *#   #    $  " *# $ #   # "       {     # !  research, many known inhibitory receptors are still not fully characterized on haematopoetic cells in general and on mast cells in particular. Therefore, there is a need for continued research to better characterize the existing inhibitory receptors, for example by studying their possible interactions with different activating receptors and discovering some of their unknown ligands. In addition, an effort should be made to identify other new inhibitory receptors on human mast cells.

REFERENCES 1. Isakov N, ITIMs and ITAMs. The Yin and Yang of antigen and Fc receptor-linked signaling machinery. Immunol Res 1997; 16(1):85-100. 2. Barrow AD, Trowsdale J. You say ITAM and I say ITIM, let’s call the whole thing off: the ambiguity of immunoreceptor signalling. Eur J Immunol 2006; 36(7):1646-53. Œ ‘  *   $^#     <  ^{   $  Pharmacol 1998; 31(1):9-18. 4. Nicholson DW. Caspase structure, proteolytic substrates and function during apoptotic cell death. Cell Death Differ 1999; 6(11):1028-42. 5. Vaux DL, Korsmeyer SJ. Cell death in development. Cell 1999; 96(2):245-54. 6. Guicciardi ME, Gores GJ. Life and death by death receptors. FASEB J 2009; 23(6):1625-37. 7. Karra L et al. Are we ready to downregulate mast cells? Curr Opin Immunol 2009; 21(6):708-14. —  # @~ @# ~ ž < *= ‚! { $ ^{    ^ $ lipid mediators. Nat Rev Immunol 2008; 8(5):349-61. 9. Bachelet I, Levi-Schaffer F, Mekori YA. Mast cells: not only in allergy. Immunol Allergy Clin North Am 2006; 26(3):407-25. 10. Piliponsky AM et al. Non-IgE-dependent activation of human lung- and cord blood-derived mast cells is induced by eosinophil major basic protein and modulated by the membrane form of stem cell factor. Blood 2003; 101(5):1898-904. 11. Bischoff SC. Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nature Reviews in Immunology 2007; 7(2):93-104. 12. Rocha-de-Souza CM et al. Human mast cell activation by Staphylococcus aureus: interleukin-8 and tumor necrosis factor alpha release and the role of Toll-like receptor 2 and CD48 molecules. Infect Immun 2008; 76(10):4489-97.

DOWN-REGULATION OF MAST CELL RESPONSES THROUGH ITIM

157

13. Lebbink RJ, Meyaard L. Non-MHC ligands for inhibitory immune receptors: novel insights and implications for immune regulation. Mol Immunol 2007; 44(9):2153-64. 14. Daëron M, Jaeger S, Du Pasquier L et al. Immunoreceptor tyrosine-based inhibition motifs: a quest in the past and future. Immunol Rev 2008; 224:11-43. 15. Cantoni C, Bottino C, Augugliaro R et al. Molecular and functional characterization of IRp60, R a member of the immunoglobulin superfamily that functions as an inhibitory receptor in human NK K cells. Eur J Immunol 1999; 29(10):3148-59. 16. Abramson J, Rozenblum G, Pecht I. Stable knockdown of MAFA expression in RBL-2H3 cells by siRN R A retrovirus-delivery system. Immunol Lett 2004; 92(1-2):179-84. 17. Drickamer K, Taylor ME. Biology of animal lectins. Annu Rev Cell Biol 1993; 9:237-64. 18. Izawa K, Kitaura J, Yamanishi Y et al. An activating and inhibitory signal from an inhibitory receptor LMIR3/CLM-1: LMIR3 augments lipopolysaccharide response through association with FcRgamma in mast cells. J Immunol 2009; 183(2):925-36. 19. Eissmann P, Beauchamp L, Wooters J et al. Molecular basis for positive and negative signaling by the natural killer cell receptor 2B4 (CD244). Blood 2005; 105(12):4722-9. _• \‹$ † ƒ$ \  $ š      #‚   #   #   { $   ‚ *  $ _••– __˜™Œ^’_ 21. Hamerman JA, Tchao NK, Lowell CA et al. Enhanced Toll-like receptor responses in the absence of signaling adaptor DAP12. Nat Immunol 2005; 6(6):579-86. 22. Katz HR. Inhibitory receptors and allergy. Curr Opin Immunol 2002; 14(6):698-704. 23. Benhamou M, Bonnerot C, Fridman WH et al. Molecular heterogeneity of murine mast cell Fc gamma receptors. J Immunol 1990; 144(8):3071-7. _’ ²    ˆ ƒ$    ‚$  ##^  =  ^   !  $  %^     > @ ! €€– €˜_™””^— 25. Fong DC, Malbec O, Arock M et al. Selective in vivo recruitment of the phosphatidylinositol phosphatase SHIP by phosphorylated Fc gammaRIIB during negative regulation of IgE-dependent mouse mast cell activation. Immunol Lett 1996; 54(2-3):83-91. _“ ƒ$  ‚ † $# \   &`   $  ##   !   ‚†    $ the SH2 domain-containing protein-tyrosine phosphatase SHP-1. J Biol Chem 2001; 276(9):6327-36. 27. Malbec O, Attal JP, Fridman WH et al. Negative regulation of mast cell proliferation by FcgammaRIIB. Mol Immunol 2002; 38(16-18):1295-9. 28. Crocker PR, Paulson JC, Varki A. Siglecs and their roles in the immune system. Nat Rev Immunol 2007; 7(4):255-66. 29. Yokoi H, Myers A, Matsumoto K et al. Alteration and acquisition of Siglecs during in vitro maturation of CD34 progenitors into human mast cells. Allergy 2006; 61(6):769-76. Œ• ` ~ > @ # ƒ   ^—  ! #^    # $$ superfamily. J Biol Chem 2000; 275(2):861-6. 31. Ghannadan M, Hauswirth AW, Schernthaner GH et al. Detection of novel CD antigens on the surface of human mast cells and basophils. Int Arch Allergy Immunol 2002; 127(4):299-307. Œ_ <  †# †         ^_  !  "   # mast cells and basophils. J Allergy Clin Immunol 2000; 105(6 Pt 1):1093-100. 33. Bochner BS. Siglec-8 on human eosinophils and mast cells and Siglec-F on murine eosinophils, are functionally related inhibitory receptors. Clin Exp Allergy 2009; 39(3):317-24. 34. Yokoi H, Choi OH, Hubbard W et al. Inhibition of FcepsilonRI-dependent mediator release and calcium {$"   #$     ^ $$^<  —  >   Clin Immunol 2008; 121(2):499-505 e1. 35. Tateno H, Crocker PR, Paulson JC. Mouse Siglec-F and human Siglec-8 are functionally convergent paralogs that are selectively expressed on eosinophils and recognize 6’-sulfo-sialyl Lewis X as a preferred glycan ligand. Glycobiology 2005; 15(11):1125-35. 36. Ishida A, Ohta M, Toda M et al. Mucin-induced apoptosis of monocyte-derived dendritic cells during maturation. Proteomics 2008; 8(16):3342-9. 37. Hudson SA, Bovin NV, Schnaar RL et al. Eosinophil-selective binding and proapoptotic effect in vitro of a synthetic Siglec-8 ligand, polymeric 6v-sulfated sialyl Lewis x. J Pharmacol Exp Ther 2009; 330(2):608-12. 38. Bachelet I, Munitz A, Moretta A et al. The inhibitory receptor IRp60 R (CD300a) is expressed and functional on human mast cells. J Immunol 2005; 175(12):7989-95. 39. Munitz A, Bachelet I, Eliashar R et al. The inhibitory receptor IRp60 R (CD300a) suppresses the effects of IL-5, GM-CSF and eotaxin on human peripheral blood eosinophils. Blood 2006; 107(5):1996-2003. 40. Alvarez Y, Tang X, Coligan JE et al. The CD300a (IRp60) R inhibitory receptor is rapidly up-regulated on #$ $ #    {  $  $ @Œ_ ˜‚™  signaling. Mol Immunol 2008; 45(1):253-8.

158

MAST CELL BIOLOGY

’ $ ` ˆ< * *$          #  }   %   immunoglobulin-like receptors LMIR1 and 2 derived from murine bone marrow-derived mast cells. Biochem Biophys Res Commun 2003; 307(3):719-29. ’_ †#  $}  ƒ!^#               linking IgE to CD300a. J Allergy Clin Immunol 2006; 117(6):1314-20. ’Œ $}  †#  ƒ!^#  ‚!    % {    #        < @@‚Œ  @Œ•• >   @ $ _••“– —˜™•—_^€ 44. Bustamante CD, Fledel-Alon A, Williamson S et al. Natural selection on protein-coding genes in the human genome. Nature 2005; 437(7062):1153-7. 45. Nielsen R, Bustamante C, Clark AG et al. A scan for positively selected genes in the genomes of humans and chimpanzees. PLoS Biol 2005; 3(6):e170. ’“ @ < > >$  * @   *# @Œ••   $   !$    $   leukocyte functions. Trends Immunol 2009; 30(5):209-17. 47. Li L, Yao Z. Mast cell and immune inhibitory receptors. Cell Mol Immunol 2004; 1(6):408-15. 48. Tedla N, Lee CW, Borges L et al. Differential expression of leukocyte immunoglobulin-like receptors on cord-blood-derived human mast cell progenitors and mature mast cells. J Leukoc Biol 2008; 83(2):334-43. ’€ } `‚ #  # {  $< ^<  †’   #  receptors. Immunol Rev 2007; 217:222-30. 50. Katz HR, Vivier E, Castells MC et al. Mouse mast cell gp49B1 contains two immunoreceptor tyrosine-based #   $    ! %#  %# # ##^    for IgE. Proc Natl Acad Sci USA 1996; 93(20):10809-14. 51. Lu-Kuo K JM, Joyal DM, Austen KF et al. gp49B1 inhibits IgE-initiated mast cell activation through both immunoreceptor tyrosine-based inhibitory motifs, recruitment of src homology 2 domain-containing phosphatase-1 and suppression of early and late calcium mobilization. J Biol Chem 1999; 274(9):5791-6. 52. Daheshia M, Friend DS, Grusby MJ et al. Increased severity of local and systemic anaphylactic reactions  ’€†^   > ="  _••– €’˜_™__”^Œ’ 53. Zhou JS, Friend DS,Feldweg AM et al. Prevention of lipopolysaccharide-induced microangiopathy by gp49B1: evidence for an important role for gp49B1 expression on neutrophils. J Exp Med 2003; 198(8):1243-51. 54. Castells MC, Klickstein LB, Hassani K et al. gp49B1-alpha(v)beta3 interaction inhibits antigen-induced mast cell activation. Nat Immunol 2001; 2(5):436-42. 55. Chen CC, Hurez V, Brockenbrough JS etal. Paternal monoallelic expression of the paired immunoglobulin-like receptors PIR-A and PIR-B. Proc Natl Acad Sci USA 1999; 96(12):6868-72. 56. Uehara T, Bléry M, Kang DW et al. Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B. J Clin Invest 2001; 108(7):1041-50. 57. Bléry M, Kubagawa K H, Chen CC et al. The paired Ig-like receptor PIR-B is an inhibitory receptor that recruits the protein-tyrosine phosphatase SHP-1. Proc Natl Acad Sci USA 1998; 95(5):2446-51. 58. Nakamura A, Kobayashi E, Takai T. Exacerbated graft-versus-host disease in Pirb-/- mice. Nat Immunol 2004; 5(6):623-9. 59. Verbrugge A, Ruiter R Td T, Clevers H et al. Differential contribution of the immunoreceptor tyrosine-based inhibitory motifs of human leukocyte-associated Ig-like receptor-1 to inhibitory function and phosphatase recruitment. Int Immunol 2003; 15(11):1349-58. 60. Meyaard L. The inhibitory collagen receptor LAIR-1 (CD305). J Leukoc Biol 2008; 83(4):799-803. 61. Meyaard L, Adema GJ, Chang C et al. LAIR-1, a novel inhibitory receptor expressed on human mononuclear leukocytes. Immunity 1997; 7(2):283-90. 62. Fournier N, Chalus L, Durand I et al. FDF03, a novel inhibitory receptor of the immunoglobulin superfamily, is expressed by human dendritic and myeloid cells. J Immunol 2000; 165(3):1197-209. 63. Verbrugge A, Rijkers ES, de Ruiter R T et al. Leukocyte-associated Ig-like receptor-1 has SH2 domain-containing phosphatase-independent function and recruits C-terminal Src kinase. Eur J Immunol 2006; 36(1):190-8. “’  #  \# >`      $  $  @_••‚^ #   cell activation. J Leukoc Biol 2006; 79(2):363-8. 65. Shiratori I, Yamaguchi M, Suzukawa M et al. Down-regulation of basophil function by human CD200 and human herpesvirus-8 CD200. J Immunol 2005; 175(7):4441-9. 66. Zhang S, Cherwinski H, Sedgwick JD et al. Molecular mechanisms of CD200 inhibition of mast cell activation. J Immunol 2004; 173(11):6786-93. 67. Cherwinski HM, Murphy CA, Joyce BL et al. The CD200 receptor is a novel and potent regulator of murine and human mast cell function. J Immunol 2005; 174(3):1348-56. 68. Abramson J, Pecht I. Regulation of the mast cell response to the type 1 Fc epsilon receptor. Immunol Rev 2007; 217:231-54. 69. Pinter E, Barreuther M, Lu T et al. Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) tyrosine phosphorylation state changes during vasculogenesis in the murine conceptus. Am J Pathol 1997; 150(5):1523-30.

DOWN-REGULATION OF MAST CELL RESPONSES THROUGH ITIM

159

70. Jackson DE. The unfolding tale of PECAM-1. FEBS Lett 2003; 540(1-3):7-14. ” %  % &  # >      # ##   =  $  #   phosphorylation of the surface adhesion protein PECAM-1 (CD31). J Biol Chem 1997; 272(20):13412-8. 72. Hua CT, Gamble JR, Vadas MA et al. Recruitment and activation of SHP-1 protein-tyrosine phosphatase  #$  #  # $^ ˜\=@ ^™    $  tyrosine-based inhibitory motiff like binding motifs and substrates. J Biol Chem 1998; 273(43):28332-40. 73. Piali L, Hammel P, Uherek C et al. CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium. J Cell Biol 1995; 130(2):451-60. 74. Deaglio S, Morra M, Mallone R et al. Human CD38 (ADP-ribosyl cyclase) is a counter-receptor of CD31, an Ig superfamily member. J Immunol 1998; 160(1):395-402. 75. Abramson J, Xu R, Pecht I. An unusual inhibitory receptor—the mast cell function-associated antigen (MAFA). Mol Immunol 2002; 38(16-18):1307-13. 76. Ortega Soto E, Pecht I. A monoclonal antibody that inhibits secretion from rat basophilic leukemia cells and binds to a novel membrane component. J Immunol 1988; 141(12):4324-32. 77. Abramson J, Licht A, Pecht I. Selective inhibition of the Fc epsilon RI-induced de novo synthesis of mediators by an inhibitory receptor. EMBO J 2006; 25(2):323-34. 78. Xu R, Abramson J, Fridkin M et al. SH2 domain-containing inositol polyphosphate 5v-phosphatase is the main mediator of the inhibitory action of the mast cell function-associated antigen. J Immunol 2001; 167(11):6394-402. ”€ ³ @ †$  #% ˆ   @$     =^#      # murine killer cell lectin-like receptor G1. J Immunol 2006; 176(3):1311-5. 80. Ito M, Maruyama T, Saito N et al. Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK K cell cytotoxicity. J Exp Med 2006; 203(2):289-95. 81. Tessmer MS, Fugere C, Stevenaert F et al. KLRG1 binds cadherins and preferentially associates with SHIP-1. Int Immunol 2007; 19(4):391-400. 82. Schwartzkopff S, Gründemann C, Schweier O et al. Tumor-associated E-cadherin mutations affect binding to the killer cell lectin-like receptor G1 in humans. J Immunol 2007; 179(2):1022-9. 83. Wu HJ, Bondada S. CD72, a coreceptor with both positive and negative effects on B lymphocyte development and function. J Clin Immunol 2009; 29(1):12-21. —’ ž `  ~< = \  >‚     #$   #$  # $ ƒ^_ B-cell differentiation antigen and sequence of the corresponding cDNA. J Immunol 1990; 144(12):4870-7. 85. Grupp SA, Harmony JA, Baluyut AR R et al. Early events in B-cell activation: anti-Lyb2, but not BSF-1, induces a phosphatidylinositol response in murine B cells. Cell Immunol 1987; 110(1):131-9. 86. Polla BS et al. Differential induction of class II gene expression in murine preB-cell lines by B-cell stimulatory factor-1 and by antibodies to B-cell surface antigens. J Mol Cell Immunol 1988; 3(6):363-73. 87. Subbarao B, Morris J, Baluyut AR. Properties of anti-Lyb-2-mediated B-cell activation and the relationship between Lyb-2 molecules and receptors for B-cell stimulatory factor-1 on murine B-lymphocytes. Cell Immunol 1988; 112(2):329-42. —— $#  & @ ƒ       @”_   #   #  ž semaphorin CD100: a novel mechanism for regulating B-cell signaling. Immunity 2000; 13(5):621-31. 89. Kataoka TR, Kumanogoh K A, Bandara G et al. CD72 negatively regulates KIT-mediated responses in human mast cells. J Immunol 184(5):2468-75. €• * &   *#         #$ =  #$ ‚ # inhibits antigen-induced histamine release by human mast cells and basophils. Allergy 2004; 59(7):772-80. 91. Zhu D, Kepley CL, Zhang M et al. A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation. Nat Med 2002; 8(5):518-21. €_  #   @ƒ *  *   #   ^  = !   #$  Fcgamma-Fcepsilon bifunctional fusion protein. J Allergy Clin Immunol 2004; 114(2):321-7.

SECTION IV

MAST CELLS IN HEALTH AND DISEASE

CHAPTER R 10 THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY Christopher P. Shelburne1   ~  #|,2 1

Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA; 2Departments of Pathology, Immunology, and Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA, and Program in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore |   3 % & &QQV " 

Abstract:

Mast cells (MCs) were once considered only as effector cells in pathogenic IgE- and IgG-mediated responses such as allergy. However, developments over the last 15   #! $ # @ #! !!  !        #   !!  # !   {     $   *#  ^{  !  #         initiating innate responses in many different models of infection and more recently,  # !  ! $  %   #   # led to the discovery that small MC-activating compounds can behave as adjuvants in vaccine formulations. Thus, our continued understanding of the MC in the context of infectious disease is likely to not only expand our scope of the MC in the normal processes of immunity, but provide new therapeutic targets to combat disease.

INTRODUCTION The vertebrate immune system has evolved two main response systems designed to        *#     $    !    #    !    #1 The second response, called adaptive immunity, is a slower, precisely targeted response that develops over a period of a few days to weeks, resulting in life-long immunological memory against the invading pathogen.2 The initiation of both of these responses during infection   $   $    ^{     #    whose function is to activate and recruit various effector cells, such as neutrophils that

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 162

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

163

dictate innate clearance of the pathogen and dendritic cells that initiate adaptive immune responses.3 ˆ   #      # !  #  ^{  environment is the MC. MCs are granulated, tissue-resident leukocytes best known for their   # {  ‹$ $#   4-7 However, numerous studies ! #     #! # $    # @  #      responder in the innate defense against bacterial infections.8,9 Recently, this protective function has been extended to their role in the induction of adaptive immune responses $   *# $ # @ #! !!   !  ^{  signals critical to the induction of both innate and adaptive immune responses during infection. In this chapter, we will evaluate the literature supporting three arguments that $ !   @        $    #  of adaptive immunity. These arguments include their conserved localization in tissues throughout evolution, their diverse expression of pattern recognition receptors and their $‹$        ^{  !   $ & %  evaluate the numerous studies demonstrating a role for the MC in the formation of both protective innate and adaptive immune responses.

THE STRA R TEGIC LOCALIZATION OF THE MAST CELL ˆ  #   !     @ # $  #     responder to infection is their positioning not only within the tissue parenchyma, but their localization within the host organism. While MCs are present in most vascularized tissues, they are generally noted to be particularly abundant in tissues that are exposed to the environment.10-13 This selective increase in MC density would presumably aid in the detection of microbes that use these sites as portals of entry into the host. While this concept has not been empirically evaluated, many studies have now demonstrated that mice containing reduced numbers of MCs or that are devoid of MCs are highly susceptible to infection.8,9,14 Another means to assess the importance of MC placement in animals is to compare their location in different vertebrate species. In mammals, MCs are readily detected in the lung, intestines, skin and urogenital tract10-13 and, in humans, have been described to reach densities of up to 500 to 4,000 per mm3 in the lungs, 7,000 to 12,000 per mm3 in skin and 20,000 per mm3 in the gastrointestinal tract.15 These densities have been noted to increase in the skin of humans that is not covered by clothing, suggesting that regions that experience continual environmental exposure respond by either recruiting more MCs or inducing their local proliferation.16 Similarly, in lower order vertebrates, including amphibians, reptiles and birds, MC analogues that share structural and functional characteristics with their mammalian counterparts are found in the mucosa of the skin and intestines.17-19      #  $    and erythrinids, MCs have been found in the skin, intestines and in the gills, a site that is continually exposed to pathogenic and environmental insult.20-25 Another common feature of MC placement in the tissues of different vertebrate species is that they are typically found deep beneath the epithelium in the mucosa, in close proximity to the blood vasculature. This selective placement of MCs near the !$$   $  ##   @  ! ^{   $ $ histamine and TNF, has near instantaneous effects on the local endothelium. In rodents, changes that MC-derived products may exert on local endothelium include increased leakiness between cells to promote edema and the induction of proteins that signal innate

164

MAST CELL BIOLOGY

effector cells, such as neutrophils or dendritic cells, to enter the tissue site to promote their antimicrobial functions. For instance, recent studies have found that MC release of tumor necrosis factor-_ (TNF-_) at the local site of infection induces the expression of the adhesion molecule E-selectin on the local blood vessel endothelium.26 The expression of E-selectin was required for the massive recruitment of dendritic cells into the infected tissue site and the subsequent induction of antibacterial humoral immunity.26 Similarly, in

# @ #!      " %#   #  # $#$ the mucosa of the intestines, skin and the loose connective tissue of the gill arch.21,27-30  %#  ‘    "  # $  @  $ #    %# #  $  $ #  # { $  Overall, the striking commonalities in different vertebrate species concerning strategic MC density and placement within tissues, suggests that common mechanisms have been  # $#$ !$  $  # @      $  

THE MAST CELL MICROBIAL SENSORY ARRA R Y     @  #!           MCs must express a diverse sensory array of receptors that facilitate the recognition of pathogens, pathogen products or host components activated in the presence of pathogens such as complement. Moreover, these sensors must translate these signals into   ^{  " @ #!   <%  $   $  bind both Salmonella and the helminth Schistosoma mansoni.31,32 MCs were also known to directly engage enterobacteria such as E. coli by binding FimH, the protein expressed  #   $ *    !     33 We now know that this protein is CD48 and engagement of E. coli through this receptor induces

@    ^{  < $#  *~34 More recent studies have demonstrated that MCs can express a number of genetically encoded pattern recognition receptors (PAMPS) that recognize and bind conserved components of different pathogens such as lipopolysaccharide (LPS). These PAMPs include Toll-like receptors (TLRs) 1, 2, 3, 4, 5, 6, 7 and 9, whose expression appears to vary according to the MC model.35-37     *ƒ‚ "       <  ! @   marrow derived MCs revealed that fetal-skin derived MCs express much higher levels of TLRs 3, 7 and 9 than bone marrow derived MCs. Moreover, several studies examining # *ƒ‚ "      #$ $$  @ #! !      TLR R expression that collectively include TLRs 1, 2, 3, 4, 5, 6, 7 and 9. Not surprisingly, engagement of TLRs with their ligands on cultured MCs induces the secretion of copious amounts of cytokines35,36 that differ based on the TLRs that are activated. For instance, Kulka et al found that stimulation of human MCs with the TLR3 ligand poly I:C resulted K in the production of IFN-_, but not TNF, IL-1`, IL-5 or GM-CSF.35 By contrast, treatment of human MCs with several TLR2 activators induced substantial IL-1` and GM-CSF responses in the absence of a Type 1 IFN response.38 Treatment of bone- marrow derived MCs with the TLR9 agonist CpGs resulted in the release of TNF, but not GM-CSF, IFN-a, IL-4 or IL-12.37 Recent studies have also found that stimulation of peritoneal derived MCs from mice with different TLR2 ligands can result in the production of differential cytokine responses. For instance, stimulation of peritoneal MCs with lipoteichoic acid (LTA) resulted in the production of IL-6, IL-1 and TNF-_, whereas treatment with macrophage activating ^_ ˜ ƒ\^_™ $  #  $  # ^{  <

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

165

IL-10.39 TLRs on MCs are also known to be functionally relevant to different primary #  !!  "   @    *ƒ‚’ %  $   less able to survive in the cecal ligation and puncture (CLP) model of innate immunity than mice containing wild-type MCs.40,41 This failure was linked to the inability of these mice to recruit neutrophils into the peritoneal cavity.40,41   @    *ƒ‚Œ %  $   $     $ *   ‘  # *ƒ‚Œ  polyI:C42    @    *ƒ‚” %  $    $ dendritic cell migration to lymph nodes after injection of the TLR7 agonist imiquimod.43 Cultured human MCs have also been found to express dectin-1, a PAMP involved in the recognition of fungal pathogens. In this model, exposure of human MCs to yeast zymosan induced the production of the cysteinyl leukotrienes (LT)-C4 and LTB4, which was inhibited in the presence of a dectin-1 inhibitor.44 Finally, MCs have recently been found to express # ~ƒ‚\Œ {   \ \ "   #   45 MCs expressing an allele of NLRP3 responsible for cryopyrin-associated periodic syndrome (CAPS) were found to spontaneously release IL-1` that induced neutrophil recruitment and vascular leakage associated with the CAPS disease.45 MCs are also activated by the adjuvant compound alum,46 which has also been suggested to activate the NLRP3 { #%!  # < #    !$ Studies have also evaluated the ability of MCs to engage humoral factors which comprise important components of the innate immune system. These include components of the complement cascade, LPS binding protein, C-reactive protein, pentraxins and collectins. Like PAMPs, such as TLRs, these circulating innate immunoproteins, which are evolutionarily conserved in both vertebrate and invertebrate species, are involved in both sensing of microbes and triggering of mechanisms to facilitate clearance of the infection. MCs in rodents and humans are now known to express several complement receptors including the C3aR,47 C5aR,48 CR2, CR449 as well as the _2`1 integrin which is a receptor for C1q.50 In vitro, engagement of C3a was found to induce MC activation and chemokine production from human MCs.51,52 In a murine model of septic peritonitis, @Œ^   %  $  $ @^ $ #  $ ‹$  for bacterial clearance.53 Repletion of these mice with C3 restored MC-dependent effector functions.53 Subsequent studies found that C3 likely engages the CR2 receptor on MCs, as   @    @‚_ %  $         49 MCs are also able to bind several collectins, including mannose-binding lectin (MBL), surfactant protein A (Sp-A) and C1q complement protein through the _2`1 integrin. This interaction is required for MC- dependent responses against Listeria monocytogenes and the yeast cell wall component zymosan.50 MCs from multiple sources have also been shown to respond to the presence of peptides released from epithelial, endothelial or neural sources during infection or insult to the host. These include bradykinin,54,55 substance P,55,56 somatostatin,55,57 adrenocorticotropic hormone (ACTH),54,55 calcitonin gene-related peptide (CGRP), R 55,58 corticotrophin-releasing 59 55,60 hormone (CRH), vasoactive intestinal peptide (VIP), neurotensin,61,62 neuromedin 63 64 U and peptide antibiotics such as `-defensins and LL-37.65-67 Many of these peptides evoke release of MC mediators including histamine,59,64,66-68 cytokines and chemokines.65-67,69,70 Functionally, neuromedin U has been implicated in mediating the

@^ {  !    $§ ‘$!      {63 Responsiveness of MCs to these peptides is dependent upon their tissue localization, as MCs derived from the skin or serosal sources are generally more responsive to these peptides than those derived from the intestines.55 Interestingly, MCs

166

MAST CELL BIOLOGY

do not always appear to express canonical receptors for each of these peptides. With the "  # $   $  '   %## #!     MCs, many of these peptide agonists of MCs have been suggested to stimulate MCs by crossing the cell membrane to directly activate pertussis toxin sensitive G (Gi_2 and Gi_3) proteins.71 Alternatively, these peptides may act through Mas related gene (MRG)-X1 and X2 receptors on human cultured MCs or their rat analogues MRGB3 or MRGB8     @ ‚     %     ^  $ receptors that can bind multiple cationic compounds and have been suggested to mediate the MC-activating properties of several host-derived peptides, including substance P, somatostatin and vasoactive intestinal peptide.71 Overall, these many observations clearly indicate that MCs from rodent or human sources have the capacity to directly respond to the presence of diverse pathogens, their products, or indirectly through host components activated by the presence of pathogens or pathogen products. A comprehensive summary of bacteria, viruses, protozoan parasites, their products or host-derived compounds demonstrated to activate MCs in vitro, as well as known receptors expressed by MCs for some of these ligands are listed in Tables 1 and 2.

Table 1. Ligands that induce MC activation in vitro Stimulus Bacteria E. coli K. pnuemoniae K. oxytoca S. aureus S. epidermidis S. cohnii P. vulgaris H. pylori M. tuberculosis Z  ' [ S. pneumonia S. viridians S. agalactiae B. catarrhalis B. pertussis S. typhimurium B. burgdorferi M. hominis U. urealyticum B. capillosus A. naeslundii L. fermentum E. cloacae P. aeriginosa

Preformedd









?

/







?











/-

Cytokines/ Chemokines





? ?





? ?

?

? ? ? ? ? ?

References 148-151 148-149,151 149 149-150,152-153 149-150 150 149 154-157 158 152-153 82,159 152 150 152-153 160 31 161-162 150 150 150 150 150 149-150 163-165 continued on next page

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

167

Table 1. Continued Stimulus

Preformed d

Cytokines/ Chemokines



?

Viruses Sendai virus Dengue virus Reovirus HIV {$} ž $ Parasites S. mansoni > = L. infantans L. brasiliensis T. spiralis TSL-1 antigen Toxins T T Toxin A ( <)  Cholera toxin (Vibrio cholera) a VacA (Cytotoxin from H. Pylori) Hemolysin (E. ( coli) Hemolysin (S. ( marcescenss) Hemolysin ((A. hydrophilia a) Hemolysin (L. ( monocytogeness) Pertussis T Toxin (B. pertussiss) Listeriolysin O Streptococcal pyrogenic exotoxin B Streptolysin O Staphylococcal enterotoxin B TLR R agonists PGN PGN Lipotechoic Acid MALP-2 Lipophosphoglycan Lipoarabinomannan dsRNA, poly I:C LPS Flagellin ssRNA, imiquimod, R848 CpG DNA Zymosan Host derived Products C3a C5a C1q Collectins Substance P







?







?

-



















References 166 167-168 169 170-171 172 173 174 174 175 176

? ? ?

177 178-179 180 181 181-182 181 181 160 183 184 185 186

?

















40 38,90,92,187-188 39 39 189 188 35,90,169 40,90,92,187 35 90 37,90,170 44















32,51-52,190-193 51,191,193-197 50,198 50,199 55,64,69-70,200-206













/-

continued on next page

168

MAST CELL BIOLOGY

Table 1. Continued Stimulus

Preformedd

Somatostatin Vasoactive intestinal peptide Neurotensin ` -defensins LL-37 Endothelin-1 Bradykinin Corticotropin releasing hormone











Cytokines/ Chemokines ?

?





?

References 55,203-204,206 55,70,203-204,206 55,61,200,205-209 64-66 65-67 113,210-211 55,205 59,212

Pre-formed indicate MC mediators that are released from the MC granule such as histamine or ` -hexosaminidase. Secondary mediators indicate mediators that are released after de-granulation, such as cytokines, chemokines or eicosanoids.

indicates a positive response. < indicates no response was detected. /< indicates that both positive and negative responses have been reported. ? indicates that no such response was evaluated.

Table 2. The microbial sensory array of MCs Receptorr CD48 TLR1 TLR2

TLR3 TLR4 R TLR5 TLR6 TLR7 R TLR9 Dectin-1 C3aR C5aR CR2 CR3 _2`1 integrin ETA NT receptor NMU-R1 MRG R -receptors

Ligand FimH Lipopeptide Peptidoglycan Lipotechoic acid MALP-2 Lipophosphoglycan Lipoarabinomannan dsRNA/Poly I:C Lipopolysaccharide Flagellin Peptidoglycan, zymosan ssRNA/Imiquimod Bacterial DNA/CpGs Zymosan C3a C5a C4b, C3b, iC3b, C3d C4b, C3b, iC3b, C3d C1q, collectins, SP-A, zymosan ET-1 Neurotensin Neuromedin U Substance P, somatostatin, VIP

References 158,213 38 38,40,90,92,187-188 39 39 189 188 35,90,169 40,92 35 38,40,92,187 43,90 37,90,170 44 32,51-52,191-193 194-196,214 49,190 14 50 113,210-211 200 63 71

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

169

THE PRO-INFLAMMATORY ARSENAL OF THE MAST CELL One of the most compelling arguments for the notion that MCs have evolved as

   $    # #   $‹$ ‹$  #    #  $ $  ^{    # #! #  # !$         % $ {   *#    ^{  $   @ #!  !  % ‘  based on whether they are prestored or de-novo synthesized. Pre-stored compounds are packaged into cytoplasmic MC granules, which are the dominant feature of MCs in all vertebrates. These granules are enveloped by a membrane and are organized around a highly sulfated glycosylaminoglycan such as heparin or chondroitin sulphate.72,73 Pre-stored components in rodent and human MC granules include soluble biogenic amines such as histamine and serotonin.74,75 Numerous enzymes and proteases such as chymases, tryptases and carboxypeptidase A,76-78 and cytokines such as TNF-_,79 are also packaged within the granule and remain associated with the glycosylaminoglycan matrix after release of the granule into the tissue.80,81 Other prestored compounds of note include antimicrobial peptides such as cathelcidin.82 De novo synthesized mediators produced by rodent and human MCs include arachidonic acid-derived metabolites generated through the cyclooxygenase pathways such as prostaglandin (PG)-D2 or the lipoxygenase pathways that yield leukotrienes such as LT-C4.76 MCs also secrete an amazing array of cytokines and chemokines, including IL-1,40,83,84 IL-3,83,85,86 IL-4,40,83,85 IL-5,87-89 IL-6,40,83,87,90 IL-8,89 IL-9,87,91 IL-10,92 IL-13,40,89,92 GM-CSF,89 TGF-`,93 CCL1,94 CCL2,94 CCL3,89,90 CCL4,94 CCL590 and CXCL2.90 Many of these compounds have been well documented to mediate #   $ { $ {$ " !  #  $  effector cells into the tissue. In lower order vertebrates, the components of the MC granule and the secretion of de novo synthesized products are not as well characterized. However, it is generally thought that #  $             # #  $ of MCs have been noted to contain serotonin,95 lysozyme,96 carboxypeptidase A5,25 alkaline and acid phosphatases, arylsulphatase, 5-nucleotidase,97,98 and potent antimicrobial peptides (called piscidins).23 Notably, histamine has not generally been detected in the granules of

#  # @ %##   #   #$#  #!    # @  reptiles. However, recent studies have demonstrated that histamine is present in the MC granules of the Perciformes order, considered the most evolutionarily advanced order of

# *# $ # #    #    ^{  $  @ granules may have occurred as a separate evolutionary event. Functionally, activation of

@  %   !   #   %# {  !  #  ‘  !    # $#   99 or brown trout28  @  $ " %  !   {  100-102 ‘  # %# # @  $ $ ’——•  !  in perfused brachial vascular beds of rainbow trout.103 *#       mammalian MCs undergo rapid exocytic responses to compound 48/80, resulting in the $   $  ^{  74,104 This point suggests that, not only   @  % !   @    {   but that they may also be hard-wired to respond to similar secretagogues. *#   @  $  #   ^{   $  microbial challenge is one of the principal ways in which MCs are thought to provoke the #    { ="  # @  $ $  # $

170

MAST CELL BIOLOGY

    #     ^{   $ $#  #   the granule itself is released intact into the local tissue, suggesting that its associated mediators such as TNF-_ and many enzymes are retained within its matrix. This might ensure # #  ^{     $$  !      "  #  %# # $  ‚ $   #!   # ## demonstrating that the MC granule conveys MC-derived TNF-_ to distal sites such as the   #  $  #   ^{    $^ 105 Intriguingly, MC granule exocytosis has been demonstrated to be somewhat variable   # $$ *#  !  { # # ^ $ ! is not an all or nothing process, but rather an exquisitely tuned response that releases an    $   ^{        # $$ *#     $       =  ˜¡RI)-mediated crosslinking on the MC surface. In this model, MC granules begin to swell within minutes, followed by fusion of individual granules into cytoplasmic channels. The channels then fused with the plasma membrane resulting in an explosive expulsion of the granular material.106,107 `%!   # "  "$   *^   E. coli, MC de-granulation occurs as a gradual exocytic event that takes an hour to complete.108 Similarly, injection of E. coli into footpad tissues results in a gradual release of granule components from MCs over a four hour period, as opposed to the near immediate release of MC granules upon injection of the MC secretagogue compound 48/80.109 The slow release of MC  $ $    #$#  {      $    degranulation, which involves the slow release of granular material as the granule fuses with the cellular membrane followed by re-internalization of the MC granule. Interestingly, many bacterial products, including TLR R ligands such as LPS or polyI:C, or toxins such as cholera toxin, do not induce MC degranulation.35-37 Rather, as noted, exposure of MCs to these bacterial components results in the release of copious amounts of cytokines.35-37

MAST CELL MODULATION OF INNATE RESPONSES TO INFECTION Overall, these observations suggest that MCs are well positioned and armed to be a      #   { ‹$   # $    adaptive immune responses during infection. Proof of this hypothesis, however, requires the use of in vivo models that facilitate the direct analysis of MCs and their impact on these responses during infection. Fortunately, certain mutant mice virtually lacking

@   ! <    !$ #    $  @ *# @^  $  $ # &††“^W/KitWv (W/Wv) mouse, a heterozygous mutant that bears a deleted kitt allele and a mutant kitt allele with defective kinase activity.110 Recently, the KitWsh/KitWsh mice have been described as an alternative model to the W/Wv strains. These mice are preferable as they do not manifest some of the !     # &&!     $  $    111 † ‹$      % @^    #  %^    #  } #   @^   # #!  ! $ %# $$  @ #    !!  $  @   {     !$110  #  $ # "    @  # $   immunity during infection in vivo were performed by Malaviya et al and Echtenacher et al in mice.9,112 Using an infectious model of bacterial peritonitis, Malaviya et al found

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

171

that injection of FimH-expressing enterobacteria into the peritoneal cavity of W/Wv mice resulted in a near 80% mortality rate, as opposed to a 0% mortality rate in wild-type controls.112 Repletion of the peritoneal cavity of W/Wv mice with cultured MCs restored the ability of these mice to resist enterobacteria induced mortality.112 *#    that the differences observed in susceptibility to infection were due to the absence of MCs and not to other abnormalities that may exist in these mice.112 MCs were found to protect mice from enterobacterial challenge by recruiting neutrophils into the infected   @^   "#  ^ $  #    $ #  #   !    @^$   112 *#  %  associated with the release of large amounts of local TNF-_, which was required for the recruitment of the neutrophils. Similar data, obtained using the cecal ligation and puncture (CLP) model of acute peritonitis, also found that MC-induced recruitment of neutrophils is important for protection against bacterial infection.9 *#     #   # @            # ability to recruit neutrophils into the site of infection. Subsequent studies have expanded our understanding of how MCs affect innate      %# $ #  $ *#     with the limitation of the toxic effects of either host derived endogenous compounds, or exogenous mediators such as venom components that can have profound deleterious effects on host tissues. Original studies that examined this problem involved the endogenous  #^ ˜=*^™ %## #   !   %#    ## concentrations.113 MCs and MC-derived carboxypeptidase A were found to be required to limit the morbidity and mortality induced by ET-1 administration in mice, as well as the effects of ET-1 in the CLP model of peritonitis.113 Subsequently, MCs have been found to limit the toxic effects of the peptide neurotensin and the cytokine IL-6 through similar proteolytic activities.62,114 Finally, comparative work found that the sequence of ET-1 was very similar to the toxic component of the Israeli mole viper sarafotoxin 6b. Because MC proteolytic activity was required to limit the harmful effects of ET-1, it suggested that MC proteolytic activity might limit the pathogenicity of sarafotoxin as % ‘   " “  @      # #  %  •    !  #    # ! # @ $  mice. Similar data was obtained using the venom from the pit viper (Crotalinae) and the honeybee (Apis mellifera),115 suggesting that MC derived proteases can limit the toxicity of noxious substances. An additional means by which MCs affect innate immune responses is through the release of prestored peptide antibiotics. Antimicrobial peptides are distinguished by their ability to rapidly inactivate a wide range of infectious agents.116-120 These peptides are thought to act by disrupting the integrity of the microbial membrane,117 a feature thought to be mediated by two common structural characteristics shared by most antimicrobial peptides, including a net-positive charge and the propensity for folding into amphipathic secondary structures that promote their binding and insertion into microbial membranes. *#        @ %    #    23 *# #^    %   %  $   largely associated with MCs in the skin, gastrointestinal tract and the gills of nine different   #  # \     24,121 Piscidins comprise a family of 21-22 amino acid antimicrobial peptides that display a broad range of antimicrobial activity against many bacterial and protozoan pathogens.24,121-122 These include not only disease-causing    # $ # # $    $#  Candida albicans.123

172

MAST CELL BIOLOGY

Subsequently, several antimicrobial peptides, including beta defensin-4, beta defensin AMP and cathelicidin were found to be expressed in both cultured human and murine MCs.82 Similar to piscidins, these antimicrobial peptides display a wide range of antimicrobial activity. Interestingly, murine MC-derived cathelicidin was found to be important in protecting mice against infection with Group A streptococcus (GAS). For instance, ‘    # <  &#  $ %# #^  MCs exhibited GAS induced lesions of 120mm2, compared to lesions of 25mm2 in the skin of Wsh mice reconstituted with cathelicidin MCs.124 Therefore, the expression and release of antimicrobial peptides are likely an important weapon in the antimicrobial functions of the MC. Collectively, these observations have been augmented by many in vivo studies that #! !$ #   @ #        @  ^{   $     #  ˜* Œ™ *# $ #!     the idea that MCs and their products play a vital role in host defense against many different types of pathogens or toxins. There are however, several important exceptions to this   ##   @^  @^   %# #   # S. typhimurium in the peritoneal cavity did not improve the ability of these mice to resist infection. In fact, repletion of Wsh mice with MCs appeared to aggravate the severity of the disease and resulted in reduced mortality.125 In addition, the few in vivo studies that have examined a role for MCs in protection from viral infection, including infection with =#  ž $  @"< †Œ ž $ #! $ # @   results in reduced pathology of the disease.126,127 However, reconstitution of Wsh mice with MCs restores the pathogenic effects of the viral illness.126,127 In calves infected with RSV, MC activation and systemic release of their products appears to correlate with the severity of symptoms.128-129 Finally, in models where large doses of TLR R ligands are     @        # # { $  # *ƒ‚    @   $ ƒ\^$ $! or cystitis and SM360320 (a TLR7 agonist) induced hypothermia.130-132 These models offer a startling contrast to those that suggest MCs have a protective role. However, an explanation may lie in the fact that pathogens such as S. typhimurium and RSV are highly evolved to cause active disease in their respective hosts. This suggests an intriguing concept, that such pathogens may have evolved strategies to overwhelm or insidiously reprogram the MC to induce changes in the local tissue site that allow it to gain access and systemically infect the host. In addition, large, systemic amounts of TLR R ligands {    ! %#  %##  $  $ $  @     {  # $    $   shock, similar to what is observed in IgE-mediated, MC-dependent systemic anaphylaxis. *#     #     $$ @  "#    pathogenic responses.

MAST CELL MODULATION OF ACQUIRED IMMUNITY DURING INFECTION Another compelling role for the MC has recently been suggested by the realization that # {  " #  ! #        # $  # ! $  {  # #  $  ´! ^   *   ^ ^   ˜\@™ 

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

173

Table 3. In vivo models examining a role for MCs and their products in host defense Challenge Bacteria E. coli E. coli K. pneumonia K. pneumonia M. pulmonis C. rodentium S. pneumonia H. pylori Z  ' [ L. monocytogenes F. novicida S. aureus M. tuberculosis P. aeriginosa CLP CLP CLP TLR Ligands PGN PGN Poly I:C Imiquimod Viruses Newcastle Disease Virus Parasites > = > = L. amazonensis T. spiralis T. spiralis N. brasilensis P. burghei S. ratti H. longicornis Endogenous/ Exogenous Peptides $ \ Endothelin-1 Neurotensin ~$  ' Sarafatoxin 6a

Site of Challenge

Host

MC Dependent Response

Refs.

Peritoneal cavity Bladder Lungs Peritoneal cavity Lungs Intestines Skin Stomach Middle ear Peritoneal cavity Lungs Peritoneal cavity Lungs Skin Peritoneal cavity Peritoneal cavity Peritoneal cavity

Mice Mice Mice Mice Mice Mice Mice Mice Mice Mice Mice Mice Mice Mice Mice Mice Mice

Bacterial Clearance Bacterial Clearance Bacterial Clearance Bacterial Clearance Bacterial Clearance Bacterial Clearance Lesional size Bacterial Clearance Bacterial Clearance Bacterial Clearance Bacterial Clearance Bacterial Clearance Bacterial Control Bacterial Clearance Survival/Bacterial Clearance Survival/Bacterial Clearance Survival/Bacterial Clearance

8,215 216 112 217 218 219 124 220 221 222 223 214 224 225 226 40 49,227

Ear Skin Peritoneal Cavity Skin

Mice Mice Mice Mice

Lymph node hypertrophy L Vasodilation T-cell recruitment Adjuvant activity

142 40 42 43

Peritoneal Cavity

Mice

T-cell recruitment

42

Skin Skin Skin Intestines Intestines Intestines Spleen Intestines Skin

Mice Mice Mice Mice Mice Mice Mice Rat Mice

Parasite Clearance No role No role Parasite Clearance Parasite Clearance No role Parasite Clearance Parasite Clearance Parasite Clearance

228 229 230 231 232-233 234-235 236 237 238

< Peritoneal Cavity Peritoneal Cavity  Peritoneal Cavity

 Mice Mice

 Mice

{ Reduced toxicity Reduced toxicity { Reduced toxicity

_Œ€ 113 62 “Œ 115

174

MAST CELL BIOLOGY

likely dendritic cells, into local draining lymph nodes. This massive recruitment of both    #  $  # "   ^  *^ receptors on naive T cells with peptide/MHC Class II complexes on dendritic cells. In  { $ # "   $  $ ˜@’• @—• @—“™     % #    $    ! T cells.3,133 †#  #   !         *    ‹$   #   $  ^  $ #    # #  #    $   *#   #  ^{  environment created by MCs during innate responses, may well have an important role in the induction of adaptive immunity. Historically, most reports have not supported a role for MCs in the development of primary adaptive immunity.5,134-138 These data are largely derived from contact sensitization  #       @    # # # %## $ ruled out a role for the mast cell in the sensitization, or antigen presentation phase of the response.139 Similarly, several studies employing prime boost regimens for the study of #$    @^   $ # #  %     $ antibody responses, suggesting that MCs had no impact on the development of adaptive immunity.5,134-138 However, a growing body of evidence has been convincingly derived from several lines of investigation employing in vivo mouse models implying that

@  #!    # ! $   " @^  mice were observed to exhibit impaired T-cell activation in a T-cell-dependent contact sensitization model.140 The relevant experiment in this study demonstrated that transfer     @^     %# #  } *@  to allow the development of a contact sensitization response after subsequent challenge of the ear with FITC compared to controls. These data suggested that, in the absence  @   }    # *@   *^  $  and that this was correlated with reduced dendritic cell migration from the epidermis. MCs were also found to be required for the full expression of the multiple sclerosis (MS) phenotype in the experimental autoimmune encephalitis (EAE) model of MS in mice. In these studies, the induction of EAE was found to be severely impaired in both the ^$     @^    % $‹$ $ # *  in this model exhibited a poorly activated phenotype after the administration of CFA and the MOG peptide.141 In addition, several studies have demonstrated that MCs can elicit dendritic cell mobilization and accumulation in lymph nodes by crosslinking Fc¡RI or the injection of the TLR2 agonist PGN138 142 or after mosquito bite.143 Initially, the search for a role for MCs in the development of adaptive immunity during infection began with linking MC activation to the enhanced sequestration of lymphocytes in draining lymph nodes. Lymph node swelling is a common occurrence in response to infection and is the result of the recruitment and entrapment of massive numbers of lymphocytes within a 24-48 hour time period.3,133 This process is thought    # <# #   ^   # %     stimulated by specialized antigen presenting cells (APCs) that have acquired fragments of the invading pathogen.3,133 *#  $  " #  $   pathogenic strain of E. coli in a footpad model of infection in mice. Injection of E. coli rapidly elicited MC de-granulation, followed by lymph node swelling that peaked at 24 hours. This suggested that MC activation and release of their mediators, preceded lymph node swelling, raising the possibility that MC products could directly impact lymph node hypertrophy. To evaluate this, E. coli was injected into the footpads of MC

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

175

  –   % $ # E. coli failed to elicit comparable levels of lymph  % ƒ#  % $     $  @^  footpads with cultured MCs. Importantly, it was found that MCs affected the sequestration of lymphocytes in the distal lymph node by the release of TNF-_ # {%  # lymph node and provoked the increased expression of adhesion molecules known to affect lymphocytes trapping such as V-CAM-1. This “remote control” of lymph node hypertrophy by MC-released TNF-_ established that MCs could affect the entrapment of lymphocytes through communication with distal organs.109 Subsequent to this study, several groups have found that the stimulation of MCs with different MC secretagogues can induce lymph node hypertrophy, although the MC mediators required are different.138,142,143 As noted, a second important arm of the primary immune response is the recruitment of antigen-laden DCs into lymph nodes during infection. During infection, DCs acquire pathogen- derived fragments in peripheral tissues and translocate to lymph nodes, where #   !   ^  #144 Therefore, cues that affect DC homing to lymph nodes are very important to the development of primary immune responses. Interestingly, host- derived factors such as TNF-_ are known to affect DC migration.145 Because MCs produce copious amounts of TNF-_, we examined the idea that MCs might affect the migration of DCs to lymph nodes during infection. In these studies, injection of E. coli  #   @    $   < impaired DC accumulation in lymph nodes compared to wild-type controls. Reconstitution  #    @^   %# @   @   <  #  @   ! @   < $   %  $ @^ ! TNF-_ %## % $   @    % ! *#  % # $# # recruitment of DCs into the infected tissue site by increasing the expression of the adhesion marker CD62E on local blood vessel endothelium. The second was in the induction of DC chemo-attractants such as CCL21 in the lymph node. Therefore, MCs activated not only the local tissue site, but also the distal lymph node in order to increase the expression of proteins that were required for the recruitment of DCs into the tissue site followed by #    #      # $ %  # # @ %  actually involved in the induction of primary immune responses during infection. Injection of E. coli  @^   $    ^  $     %^   @^  @^   *#   @     ‹$   # !   ^  $  # $# their ability to provoke the increased recruitment of DCs and lymphocytes into lymph nodes during an infection.26 *#   #  $ # @ #   #  of the adaptive immune response by directing the mobilization of two of the important effector cells in the induction of adaptive immunity into draining lymph nodes. This led to the question, if one activated MCs in the context of a vaccine antigen, could you elicit an immune response? Subsequently, injection of the small molecule MC-activating molecule compound 48/80 (c48/80) with the vaccine antigen, protective antigen (PA) from Bacillus anthracis, in the footpad was found to generate rapid and profound anti-PA humoral responses. Injection of c48/80 plus PA failed to elicit robust responses in

@^    # @ %  ‹$   # ‘$!   ’——• ~ $     @^   %# @ %    #     % #    @ ! $ !< µ       ! $ $ $  $ # @ !  $ #!   $ ‘$! *#    $ #   

176

MAST CELL BIOLOGY

very few safe adjuvants that can induce robust mucosal immunity along mucosal surfaces. These immune responses were not only robust, but functional, in that they provided neutralization in several lethal challenge models, including an in vitro anthrax lethal toxin assay and protected mice against a lethal vaccinia virus infection in vivo.146 Subsequent studies found that c48/80 also acted as an effective adjuvant when administered though an intradermal route.147 Therefore, MC-activating compounds are an effective and novel class of vaccine adjuvant.

CONCLUSION Collectively, these numerous studies provide compelling evidence that MCs have !!          !        *# hypothesis contrasts the notion that MCs are a vestibular remnant of evolution that is only capable of causing disease such as allergy. In support of these ideas are their consistent positioning in tissues of vertebrates, extensive microbial sensory array and ability to  !  #     ^{  " #  !! $ #! found to be important for innate and adaptive immunity during infection. However, it remains important to note that MCs may also play a negative role in the pathology of some infectious diseases and it will be important to determine how this occurs. Nevertheless, #    "  # @ $ # #  #  $       @^! $ #!  !   ## ! mucosal vaccine adjuvants and certainly, continued evolution of our understanding of the MC may result in tailored vaccine adjuvants that can provide the appropriate, protective immune stimulus for different pathogens. We can also envisage manipulating the MC to stimulate innate immune responses to therapeutically manage many different types of infection. Therefore, our continued study and understanding of this fascinating cell in the context of infection will likely provide insights leading to improved therapies for not only infectious disease, but many other pathological conditions where MCs have been suggested to play a role.

ACKNOWLEDGEMENTS This work was supported by funds from the National Institutes of Health RO1-DK077159, R37-DK050814, R21-DK077307 and R01-AI 50021, the Sandler Foundation for Asthma Research and the Duke University’s CTSA grant UL1RR024128 from NCRR/NI R H.

REFERENCES 1. Medzhitov R, Janeway CA Jr. Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 1997; 9(1):4-9. 2. Gourley TS, Wherry EJ, Masopust D et al. Generation and maintenance of immunological memory. Semin Immunol 2004; 16(5):323-333. 3. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004; 5(10):987-995.

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

177

’ ƒ      $ #       $ < % $  {  arthritis. Science 2002; 297(5587):1689-1692. 5. Secor VH, Secor WE, Gutekunst CA et al. Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis. J Exp Med 2000; 191(5):813-822. 6. Chen R, Fairley JA, Zhao ML et al. Macrophages, but not T and B-lymphocytes, are critical for subepidermal     "  $$ #  #^ $ #    depends on mast cell activation. J Immunol 2002; 169(7):3987-3992. 7. Leal-Berumen I, Conlon P, Marshall JS. IL-6 production by rat peritoneal mast cells is not necessarily preceded by histamine release and can be induced by bacterial lipopolysaccharide. J Immunol 1994; 152(11):5468-5476. — ! ‚ < * ‚ =     $  $ # {$"        of infection through TNF-alpha. Nature 1996; 381(6577):77-80. 9. Echtenacher B, Mannel DN, Hultner L. Critical protective role of mast cells in a model of acute septic peritonitis. Nature 1996; 381(6577):75-77. 10. Kitamura Y. Heterogeneity of mast cells and phenotypic change between subpopulations. Annu Rev Immunol 1989; 7:59-76. 11. Kawakami T, Galli SJ. Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2002; 2(10):773-786. 12. Galli SJ, Zsebo KM, Geissler EN. The kit ligand, stem cell factor. Adv Immunol 1994; 55:1-96. 13. Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev 1997; 77(4):1033-1079. 14. Rosenkranz AR, Coxon A, Maurer M et al. Impaired mast cell development and innate immunity in Mac-1 ˜@@— @‚Œ™^   > $ €€—– “˜_™“’“Œ^“’“”  &    ^ {  #    €—€– “Œ˜“ \ _™’“^• 16. Janssens AS, Heide R, den Hollander JC et al. Mast cell distribution in normal adult skin. J Clin Pathol 2005; 58(3):285-289. 17. Chiu H, Lagunoff D. Histochemical comparison of vertebrate mast cells. Histochem J 1972; 4(2):135-144. 18. Sottovia-Filho D. Morphology and histochemistry of the mast cells of snakes. J Morphol 1974; 142(1):109-116. 19. Selye H, ed The Mast Cells. London: Butterworth; 1965. 20. Paulsen SMSB, Robertsen B. Selective staining and disintegration of intestinal eosinophilic granule cells in Atlantic salmon after intraperitoneal injection of the zinc chelator dithizone. J Fish Biol 2001; 58:768-775. 21. Murray HM LCT, Douglas SE. Immunocytochemical localization of pleurocidin to the cytoplasmic granules  #  $    # % {$  > # † _••”– ”•ŒŒ“^Œ’ __ }$ †   ƒ    #     $   # !    and degranulation. J Fish Dis 2008. _Œ #$ ' ~ => \      # ~$  _••– ’’˜“—“™_“—^_“€ 24. Silphaduang U, Colorni A, Noga EJ. Evidence for widespread distribution of piscidin antimicrobial peptides   #  ‹$ ˆ  _••“– ”_˜Œ™_’^__ _  >*   > *# =   @ "     !     # }  # providing new insight into mast cell fate determination. Blood 2008; 112(7):2969-2972. 26. Shelburne CP, Nakano H, St John AL et al. Mast cells augment adaptive immunity by orchestrating dendritic    < # $#  $ @ `   _••€– “˜’™ŒŒ^Œ’_ 27. Powell MD, Wright, GM, Burka JF. Eosinophilic granule cells in the gill of rainbow trout, Oncorhynchus mykiss: evidence of migration? J Fish Biol 1990; 37:495-497. 28. Reite OB. Mast cell/eosinophilic granule cells of salmonids: staining properties and responses to noxious  # # # $ €€”– ”“”^—’ 29. Holland JW RAF. Studies on the eosinophilic granule cells in the gills of rainbow trout, Oncorhynchus mykiss. Comp Biochem Physiol C 1998; 120:321-328. 30. Noya M, Lamas J. Response of eosinophilic granule cells of gilthead seabream (Sparus aurata, Teleostei) to bacteria and bacterial products. Cell Tissue Res 1997; 287(1):223-230. 31. Sher A, Hein A, Moser G et al. Complement receptors promote the phagocytosis of bacteria by rat peritoneal mast cells. Lab Invest 1979; 41(6):490-499. 32. Sher A, McIntyre SL. Receptors for C3 on rat peritoneal mast cells. J Immunol 1977; 119(2):722-725. 33. Malaviya R, Ross EA, MacGregor JI et al. Mast cell phagocytosis of Fim-H-expressing enterobacteria. J Immunol 1994; 152(4):1907-1914. 34. Malaviya R, Gao Z, Thankavel K et al. The mast cell tumor necrosis factor alpha response to FimH-expressing Excherichia coli is mediated by the glycosylphosphatidylinositol-anchored molecule CD48. PNAS 1999; 96:8110-8115. 35. K Kulka M, Alexopoulou L, Flavell RA et al. Activation of mast cells by double-stranded RN R A: evidence for activation through Toll-like receptor 3. J Allergy Clin Immunol 2004; 114(1):174-182.

178

MAST CELL BIOLOGY

Œ“ $# ` š ~ $ `   *ƒ‚Œ^ *ƒ‚”^  *ƒ‚€^  $   {  cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. J Immunol 2004; 173:531-541. 37. Zhu FG, Marshall JS. CpG-containing oligodeoxynucleotides induce TNF-alpha and IL-6 production but not degranulation from murine bone marrow-derived mast cells. J Leukoc Biol 2001; 69(2):253-262. 38. McCurdy JD, Olynych TJ, Maher LH et al. Cutting edge: distinct Toll-like receptor 2 activators selectively induce different classes of mediator production from human mast cells. J Immunol 2003; 170(4):1625-1629. Œ€ ^#  }  $<    $       $‹$     <  prostaglandins in response to distinct TLR2 ligands. Exp Dermatol 2009; 18(5):437-444. 40. Supajatura V, Ushio H, Nakao A et al. Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J Clin Invest 2002; 109(10):1351-1359. 41. Supajatura V, Ushio H, Nakao A et al. Protective roles of mast cells against enterobacterial infection are mediated by Toll-like receptor 4. J Immunol 2001; 167(4):2250-2256. 42. Orinska Z, Bulanova E, Budagian V et al. TLR3-induced activation of mast cells modulates CD8 T-cell recruitment. Blood 2005; 106(3):978-987. ’Œ ` ž †<  &  *        $    {    ƒ # cells and CTL responses following topical application of TLR7 ligand in mice. Blood 2007; 110(3):946-953. 44. Olynych TJ, Jakeman DL, Marshall JS. Fungal zymosan induces leukotriene production by human mast cells through a dectin-1-dependent mechanism. J Allergy Clin Immunol 2006; 118(4):837-843. 45. Nakamura Y, Kambe N, Saito M et al. Mast cells mediate neutrophil recruitment and vascular leakage through # ~ƒ‚\Œ {  #^ $   > ="  _••€– _•“˜™•Œ”^•’“ 46. McKee AS, Munks MW, MacLeod MK K et al. Alum induces innate immune responses through macrophage      $ #      ‹$   $     ‘$!    immunity. J Immunol 2009; 183(7):4403-4414. 47. Vines CM, Prossnitz ER. Mechanisms of G protein-coupled receptor-mediated degranulation. FEMS Microbiol Lett 2004; 236(1):1-6. ’— <<% >  ‚  ˆ    # $ # {    # $   #$ reaction through G alpha i2 activation that controls C5aR R and Fc receptor cooperation. J Immunol 2005; 174(5):3041-3050. 49. Gommerman JL, Oh DY, Zhou X et al. A role for CD21/CD35 and CD19 in responses to acute septic peritonitis: a potential mechanism for mast cell activation. J Immunol 2000; 165(12):6915-6921. 50. Edelson BT, Stricker TP, Li Z et al. Novel collectin/C1q receptor mediates mast cell activation and innate immunity. Blood 2006; 107(1):143-150. 51. el-Lati SG, Dahinden CA, Church MK. Complement peptides C3a- and C5a-induced mediator release from dissociated human skin mast cells. J Invest Dermatol 1994; 102(5):803-806. _ ƒ  ƒ#  >    ="   ##^  %^     @Œ  # human mast cell line, HMC-1. Eur J Immunol 1996; 26(4):753-758. 53. Prodeus AP, Zhou X, Maurer M et al. Impaired mast cell-dependent natural immunity in complement @Œ^   ~$  €€”– Œ€•˜“““™”_^” 54. Jasani B, K Kreil G, Mackler BF et al. Further studies on the structural requirements for polypeptide-mediated histamine release from rat mast cells. Biochem J 1979; 181(3):623-632. 55. Shanahan F, Denburg JA, Fox J et al. Mast cell heterogeneity: effects of neuroenteric peptides on histamine release. J Immunol 1985; 135(2):1331-1337. 56. Hagermark O, Hokfelt T, Pernow B. Flare and itch induced by substance P in human skin. J Invest Dermatol 1978; 71(4):233-235. 57. Theoharides TC, Betchaku T, Douglas WW. Somatostatin-induced histamine secretion in mast cells. Characterization of the effect. Eur J Pharmacol 1981; 69(2):127-137. 58. Piotrowski W, Foreman JC. Some effects of calcitonin gene-related peptide in human skin and on histamine release. Br J Dermatol 1986; 114(1):37-46. 59. Theoharides TC, Singh LK, Boucher W et al. Corticotropin-releasing hormone induces skin mast cell  $    !$     "    {   Endocrinology 1998; 139(1):403-413. 60. F Fjellner B, Hagermark O. Studies on pruritogenic and histamine-releasing effects of some putative peptide neurotransmitters. Acta Derm Venereol 1981; 61(3):245-250. 61. K Kurose M, Saeki K. Histamine release induced by neurotensin from rat peritoneal mast cells. Eur J Pharmacol 1981; 76(2-3):129-136. 62. Piliponsky AM, Chen CC, Nishimura T et al. Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis. Nat Med 2008; 14(4):392-398.

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

179

“Œ     * $ `   *# $  $  '   {    activation of mast cells. J Exp Med 2005; 202(2):217-224. 64. Befus AD, Mowat C, Gilchrist M et al. Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action. J Immunol 1999; 163(2):947-953. 65. Niyonsaba F, Ushio H, Hara M et al. Antimicrobial Peptides Human {beta}-Defensins and Cathelicidin LL-37 Induce the Secretion of a Pruritogenic Cytokine IL-31 by Human Mast Cells. J Immunol 2010. 66. Niyonsaba F, Someya A, Hirata M et al. Evaluation of the effects of peptide antibiotics human beta-defensins-1/-2 and LL-37 on histamine release and prostaglandin D(2) production from mast cells. Eur J Immunol 2001; 31(4):1066-1075. 67. Chen X, Niyonsaba F, Ushio H et al. Antimicrobial peptides human beta-defensin (hBD)-3 and hBD-4 activate mast cells and increase skin vascular permeability. Eur J Immunol 2007; 37(2):434-444. 68. Carraway RE, Cochrane DE, Granier C et al. Parallel secretion of endogenous 5-hydroxytryptamine and histamine from mast cells stimulated by vasoactive peptides and compound 48/80. Br J Pharmacol 1984; 81(2):227-229. 69. Ansel JC, Brown JR, Payan DG et al. Substance P selectively activates TNF-alpha gene expression in murine mast cells. J Immunol 1993; 150(10):4478-4485. 70. K Kulka M, Sheen CH, Tancowny BP et al. Neuropeptides activate human mast cell degranulation and chemokine production. Immunology 2008; 123(3):398-410. 71. Tatemoto K, Nozaki Y, Tsuda R et al. Immunoglobulin E-independent activation of mast cell is mediated by Mrg receptors. Biochem Biophys Res Commun 2006; 349(4):1322-1328. ”_    = \‘  ‚!             # ^#} enzyme. Nature 1999; 400(6746):773-776. ”Œ `$#  = & &      `     #       $   in mast cells. Nature 1999; 400(6746):769-772. 74. Riley JF. Histamine in tissue mast cells. Science 1953; 118(3064):332. 75. Kapeller-Adler R. Histamine Catabolism in Vitro and in Vivo. Fed Proc 1965;24:757-765. 76. Gurish MF, Austen KF. The diverse roles of mast cells. J Exp Med 2001; 194(1):F1-5. ”” `$ @   ! ‚ƒ ‚$  $       { > @ Immunol 1998; 18(3):169-183. 78. Compton SJ, Cairns JA, Holgate ST et al. The role of mast cell tryptase in regulating endothelial cell proliferation, cytokine release and adhesion molecule expression: tryptase induces expression of mRN R A for IL-1 beta and IL-8 and stimulates the selective release of IL-8 from human umbilical vein endothelial cells. J Immunol 1998; 161(4):1939-1946. 79. Gordon JR, Galli SJ. Mast cells as a source of both preformed and immunologically inducible TNF-alpha/ cachectin. Nature 1990; 346(6281):274-276. 80. Stevens RL, Adachi R. Protease-proteoglycan complexes of mouse and human mast cells and importance  # ^ ^#  "  {   $ $ ‚! _••”– 217:155-167. 81. Kunder K CA, St John AL, Li G et al. Mast cell-derived particles deliver peripheral signals to remote lymph nodes. J Exp Med 2009; 206(11):2455-2467. 82. Di Nardo A, Vitiello A, Gallo RL. Cutting edge: mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide. J Immunol 2003; 170(5):2274-2278. 83. Burd PR, Rogers HW, Gordon JR R et al. Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines. J Exp Med 1989; 170(1):245-257. 84. Lin TJ, Garduno R, Boudreau RT et al. Pseudomonas aeruginosa activates human mast cells to induce neutrophil transendothelial migration via mast cell-derived IL-1 alpha and beta. J Immunol 2002; 169(8):4522-4530. 85. Plaut M, Pierce JH, Watson CJ et al. Mast cell lines produce lymphokines in response to cross-linkage of Fc epsilon RI or to calcium ionophores. Nature 1989; 339(6219):64-67. 86. Tachimoto H, Ebisawa M, Hasegawa T et al. Reciprocal regulation of cultured human mast cell cytokine production by IL-4 and IFN-gamma. J Allergy Clin Immunol 2000; 106(1 Pt 1):141-149. 87. Hultner L, Kolsch S, Stassen M et al. In activated mast cells, IL-1 up-regulates the production of several Th2-related cytokines including IL-9. J Immunol 2000; 164(11):5556-5563. 88. Varadaradjalou S, Feger F, Thieblemont N et al. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells. Eur J Immunol 2003; 33:899-906. —€ &<#   $‘ š ~< *    "        ‚ <  #< upon Fc epsilon RI activation in human cultured mast cells derived from peripheral blood. Cytokine 2001; 16(4):143-152. €• $# ` š ~ $ `   *ƒ‚Œ^ *ƒ‚”^  *ƒ‚€^  $   {  cytokines and chemokines from murine connective tissue type skin-derived mast cells but not from bone marrow-derived mast cells. J Immunol 2004; 173(1):531-541.

180

MAST CELL BIOLOGY

91. Stassen M, Arnold M, Hultner L et al. Murine bone marrow-derived mast cells as potent producers of IL-9: costimulatory function of IL-10 and kit ligand in the presence of IL-1. J Immunol 2000; 164(11):5549-5555. 92. Varadaradjalou S, Feger F, Thieblemont N et al. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells. Eur J Immunol 2003; 33(4):899-906. €Œ  >@ ƒ `  >   \   $       =^ ! of mouse mast cells: role for mast cell tumor necrosis factor-alpha and transforming growth factor-beta 1. J Allergy Clin Immunol 1997; 99(1 Pt 1):113-123. 94. Nakajima T, Inagaki N, Tanaka H et al. Marked increase in CC chemokine gene expression in both human and mouse mast cell transcriptomes following Fcepsilon receptor I cross-linking: an interspecies comparison. Blood 2002; 100(12):3861-3868. 95. Dezfuli BS, Arrighi S, Domeneghini C et al. Immunohistochemical detection of neuromodulators in the intestine of Salmo trutta L. naturally infected with Cyathocephalus truncatus Pallas (Cestoda). J Fish Dis 2000; 23:265-273. 96. Sveinbjornsson BOR, Paulsen S. Immunocytochemical localization of lysozyme in intestinal eosinophilic granule cells (EGCs) of Atlantic salmon, Salmo salar L. J Fish Dis 1996; 19:349-355. 97. Powell MD, Briand HA, Wright GM et al. Ultrastructural localization of acid phosphatase in intestinal eosinophilic granule cells (EGCs) of rainbow trout (Onchorhynchus mykiss) following degranulation with capsaicin. Histol Histopathol 1992; 7:301-305. 98. Ezeasor DN, Stokoe WM. A cytochemical, light and electron microscopic study on the eosinophilic granule cells in the gut of the rainbow trout, Salmo gairdneri. J Fish Biol 1980; 17:619-634. 99. Reite OB, Evensen O. Mast cells in the swimbladder of Atlantic salmon, Salmo salar: histochemistry and responses to compound 48/80 and formalin-inactivated Aeromonas salmonicida. Dis Aquat Organ 1994; 20:95-100. 100. Vallejo AN A Jr, Ellis AE. Ultrastructural study of the response of eosinophil granule cells to Aeromonas salmonicida extracellular products and histamine liberators in rainbow trout Salmo gairdneri Richardson. Dev Comp Immunol 1989; 13(2):133-148. 101. Lamas J, Bruno DW, Santos Y et al. Eosinophilic granule cell response to intraperitoneal injection with ž  $ $   " $  $  %  $ ˆ ##$ < # # # Immunol 1991; 1:187-194. •_ ƒ >  š † $ &   ~^  $   %  $  ž  $ $ and its extracellular products (ECPs). J Fish Biol 1994; 45:839-854. 103. Matsuyama T, Iida T. Degranulation of eosinophilic granular cells with possible involvement in neutrophil      {   ! @ $ €€€– _Œ˜“™’^’” 104. Paton WD. Compound 48/80: a potent histamine liberator. Br J Pharmacol 1951; 6(3):499-508. 105. Kunder K CA, St John AL, Li G et al. Mast cell-derived particles deliver peripheral signals to remote lymph nodes. J Exp Med 2009. 106. Dvorak AM, Hammel I, Schulman ES et al. Differences in the behavior of cytoplasmic granules and lipid bodies during human lung mast cell degranulation. J Cell Biol 1984; 99(5):1678-1687. •” @$  >\ ƒ% ‚ `        #$ $    =!  solubilization of granule contents before discharge. J Cell Biol 1980; 85(2):299-312. •— ! ‚ ‚ = ><#< †      $ $      =# # coli in mice. J Clin Invest 1994; 93(4):1645-1653. 109. McLachlan JB, Hart JP, Pizzo SV et al. Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection. Nat Immunol 2003; 4(12):1199-1205. •   @# @@ \<     ^  &^# ^< $  &^#&^# mice as a model for investigating mast cell biology in vivo. Am J Pathol 2005; 167(3):835-848. 111. Wolters PJ, Mallen-St Clair J, Lewis CC et al. Tissue-selective mast cell reconstitution and differential $  "    ^  ˜&^#™˜&^#™ #  @ ="   _••– 35(1):82-88. _ ! ‚ < * ‚ =     $  $ # {$"       sites of infection through TNF-alpha [see comments]. Nature 1996; 381(6577):77-80. 113. Maurer M, Wedemeyer J, Metz M et al. Mast cells promote homeostasis by limiting endothelin-1-induced toxicity. Nature 2004; 432(7016):512-516. 114. Mallen-St Clair J, Pham CT, Villalta SA et al. Mast cell dipeptidyl peptidase I mediates survival from sepsis. J Clin Invest 2004; 113(4):628-634. 115. Metz M, Piliponsky AM, Chen CC et al. Mast cells can enhance resistance to snake and honeybee venoms. Science 2006; 313(5786):526-530. 116. Lehrer RI, Ganz T. Antimicrobial peptides in mammalian and insect host defence. Curr Opin Immunol 1999; 11(1):23-27. 117. Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002; 415(6870):389-395.

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

181

118. Hancock RE, Scott MG. The role of antimicrobial peptides in animal defenses. Proc Natl Acad Sci USA 2000; 97(16):8856-8861. 119. Ganz T.Antimicrobial polypeptides in host defense of the respiratory tract. JClin Invest 2002; 109(6):693-697. 120. Boman HG. Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scand J Immunol 1998; 48(1):15-25. _ ~ => #$ ' \  !       #  $ ~% \  2003; 16(2):87-92. __ @   '  `#   !  #     _  # ectoparasites. J Fish Dis 2008; 31(6):423-432. 123. Sung WS, Lee J, Lee DG. Fungicidal effect of piscidin on Candida albicans: pore formation in lipid vesicles and activity in fungal membranes. Biol Pharm Bull 2008; 31(10):1906-1910. 124. Di Nardo A, Yamasaki K, Dorschner RA et al. Mast cell cathelicidin antimicrobial peptide prevents invasive group A Streptococcus infection of the skin. J Immunol 2008; 180(11):7565-7573. 125. Chatterjea D, Burns-Guydish SM, Sciuto TE et al. Adoptive transfer of mast cells does not enhance the impaired survival of Kit(W)/Kit(W-v) mice in a model of low dose intraperitoneal infection with bioluminescent Salmonella typhimurium. Immunol Lett 2005; 99(1):122-129. 126. Higuchi H, Hara M, Yamamoto K et al. Mast cells play a critical role in the pathogenesis of viral myocarditis. Circulation 2008; 118(4):363-372. 127. Fairweather D, Frisancho-Kiss S, Gatewood S et al. Mast cells and innate cytokines are associated with susceptibility to autoimmune heart disease following coxsackievirus B3 infection. Autoimmunity 2004; 37(2):131-145. 128. Kimman TG, Terpstra GK, Daha MR R et al. Pathogenesis of naturally acquired bovine respiratory syncytial virus infection in calves: evidence for the involvement of complement and mast cell mediators. Am J Vet Res 1989; 50(5):694-700. 129. Jolly S, Detilleux J, Desmecht D. Extensive mast cell degranulation in bovine respiratory syncytial virus-associated paroxystic respiratory distress syndrome. Vet Immunol Immunopathol 2004; 97(3-4):125-136. 130. Li Q, Peng B, Whitcup SM et al. Endotoxin induced uveitis in the mouse: susceptibility and genetic control. Exp Eye Res 1995; 61(5):629-632. 131. Bjorling DE, Jerde TJ, Zine MJ et al. Mast cells mediate the severity of experimental cystitis in mice. J Urol 1999; 162(1):231-236. 132. Hayashi T, Cottam HB, Chan M et al. Mast cell-dependent anorexia and hypothermia induced by mucosal activation of Toll-like receptor 7. Am J Physiol Regul Integr Comp Physiol 2008; 295(1):R123-132. ŒŒ ><  # $  $ =    !! !  ^  @’ *^ $ ‚! Immunol 2001; 19:23-45. Œ’ & @   >      % !  $   #  {  an asthma model in mice. J Exp Med 2000; 192(3):455-462. Œ † $   >@ *!  >`   $     % {  ƒ^’   mice. Clin Exp Allergy 1994; 24(1):73-80. Œ“ ` *š ‚ ~ @ % \ $     ^  &&!    # Allergy Appl Immunol 1986; 80(1):85-94. 137. Kung K TT, Stelts D, Zurcher JA et al. Mast cells modulate allergic pulmonary eosinophilia in mice. Am J Respir Cell Mol Biol 1995; 12(4):404-409. 138. Jawdat DM, Albert EJ, Rowden G et al. IgE-mediated mast cell activation induces Langerhans cell migration in vivo. J Immunol 2004; 173(8):5275-5282. Œ€  > `  '‹$!  # !   ^      1984; 226(4675):710-713. 140. Bryce PJ, Miller ML, Miyajima I et al. Immune Sensitization in the Skin Is Enhanced by Antigen-Independent Effects of IgE. Immunity 2004; 20(4):381-392. 141. Gregory GD, Bickford A, Robbie-Ryan R M et al. MASTering the immune response: mast cells in autoimmunity. Novartis Found Symp 2005; 271:215-225; discussion 225-231. 142. Jawdat DM, Rowden G, Marshall JS. Mast cells have a pivotal role in TNF-independent lymph node hypertrophy and the mobilization of Langerhans cells in response to bacterial peptidoglycan. J Immunol 2006; 177(3):1755-1762. 143. Demeure CE, Brahimi K, Hacini F et al. Anopheles mosquito bites activate cutaneous mast cells leading    {    #  #  > $ _••– ”’˜”™Œ€Œ_^Œ€’• 144. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392(6673):245-252. 145. Cavanagh LL, Von Andrian UH. Travellers in many guises: the origins and destinations of dendritic cells. Immunol Cell Biol 2002; 80(5):448-462.

182

MAST CELL BIOLOGY

146. McLachlan JB, Shelburne CP, Hart JP et al. Mast cell activators: a new class of highly effective vaccine adjuvants. Nat Med 2008; 14(5):536-541. 147. McGowen AL, Hale LP, Shelburne CP et al. The mast cell activator compound 48/80 is safe and effective when used as an adjuvant for intradermal immunization with Bacillus anthracis protective antigen. Vaccine 2009; 27(27):3544-3552. ’— ! ‚ < * ‚ =     $  $ # {$"       sites of infection through TNF-alpha. Nature 1996; 381(6577):77-80. 149. Church MK, Norn S, Pao GJ et al. Non-IgE-dependent bacteria-induced histamine release from human lung and tonsillar mast cells. Clin Allergy 1987; 17(4):341-353. • † }}<¢†}}< = &  ž   {  !       J Immunopathol Pharmacol 2002; 15(3):233-238. 151. Malaviya R, Ross EA, MacGregor JI et al. Mast cell phagocytosis of FimH-expressing enterobacteria. J Immunol 1994; 152(4):1907-1914. 152. Brzezinska-Blaszczyk E, Gaik A, Czuwaj M et al. Histamine release from human pulmonary mast cells induced by bacterial antigens. Allergol Immunopathol (Madr) 1988; 16(6):375-378. Œ @ \ ƒ  ˆ  ~   `#$ {$}  #  # histamine release from human bronchoalveolar cells. Examination of patients with chronic bronchitis and controls. Apmis 1995; 103(11):806-812. 154. Masini E, Bechi P, Dei R et al. Helicobacter pylori potentiates histamine release from rat serosal mast cells induced by bile acids. Dig Dis Sci 1994; 39(7):1493-1500. 155. Bechi P, Dei R, Di Bello MG et al. Helicobacter pylori potentiates histamine release from serosal rat mast cells in vitro. Dig Dis Sci 1993; 38(5):944-949. “ š > &  `    ‚         {  ` pylori infection. J Physiol Pharmacol 1999; 50(1):17-23. 157. Lutton DA, Bamford KB, O’Loughlin B et al. Modulatory action of Helicobacter pylori on histamine release from mast cells and basophils in vitro. J Med Microbiol 1995; 42(6):386-393. 158. Munoz S, Hernandez-Pando R, Abraham SN et al. Mast cell activation by Mycobacterium tuberculosis: mediator release and role of CD48. J Immunol 2003; 170(11):5590-5596. 159. Barbuti G, Moschioni M, Censini S et al. Streptococcus pneumoniae induces mast cell degranulation. Int J Med Microbiol 2006; 296(4-5):325-329. 160. Mielcarek N, Hornquist EH, Johansson BR R et al. Interaction of Bordetella pertussis with mast cells, modulation of cytokine secretion by pertussis toxin. Cell Microbiol 2001; 3(3):181-188. 161. Talkington J, Nickell SP. Borrelia burgdorferi spirochetes induce mast cell activation and cytokine release. Infect Immun 1999; 67(3):1107-1115. 162. Talkington J, Nickell SP. Role of Fc gamma receptors in triggering host cell activation and cytokine release by Borrelia burgdorferi. Infect Immun 2001; 69(1):413-419. “Œ ƒ *> # ƒ`     !    $  @@ƒ_•   # {  protein 3alpha, by human mast cells in response to Pseudomonas aeruginosa. Infect Immun 2003; 71(1):365-373. “’ †  ' # >    $  {    ˜#  $< ™ from rat peritoneal mast cells and human granulocytes by Pseudomonas aeruginosa strains from burn patients. Infect Immun 1989; 57(7):2187-2195. 165. Friedl P, Konig B, Konig W. Effects of mucoid and nonmucoid Pseudomonas aeruginosa isolates from      {      #$  #$  $ and rat mast cells. Immunology 1992; 76(1):86-94. 166. Sugiyama K. Histamine release from rat mast cells induced by Sendai virus. Nature 1977; 270(5638):614-615. 167. Brown MG, King CA, Sherren C et al. A dominant role for FcgammaRII in antibody-enhanced dengue virus infection of human mast cells and associated CCL5 release. J Leukoc Biol 2006; 80(6):1242-1250. 168. King CA, Anderson R, Marshall JS. Dengue virus selectively induces human mast cell chemokine production. J Virol 2002; 76(16):8408-8419. 169. Burke SM, Issekutz TB, Mohan K et al. Human mast cell activation with virus-associated stimuli leads to the selective chemotaxis of natural killer cells by a CXCL8-dependent mechanism. Blood 2008; 111(12):5467-5476. 170. Sundstrom JB, Little DM, Villinger F et al. Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells. J Immunol 2004; 172(7):4391-4401. 171. Taub DD, Mikovits JA, Nilsson G et al. Alterations in mast cell function and survival following  !   %# #$ $  ! $^ # $# @@‚’ @ $ _••’– 230(2):65-80. ”_ @ \ †  ` \    #$ $ $  {$}  ! $ $ #$ bronchoalveolar cells to release histamine and leukotrienes. Agents Actions 1989; 27(1-2):107-109.

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

183

173. Catto BA, Lewis FA, Ottesen EA. Cercaria-induced histamine release: a factor in the pathogenesis of schistosome dermatitis? Am J Trop Med Hyg 1980; 29(5):886-889. 174. Bidri M, Vouldoukis I, Mossalayi MD et al. Evidence for direct interaction between mast cells and Leishmania parasites. Parasite Immunol 1997; 19(10):475-483. 175. de Oliveira MP, Lima MC, Calheiros AS et al. Leishmania (Viannia) braziliensis: human mast cell line activation induced by logarithmic and stationary promastigote derived-lysates. Exp Parasitol 2005; 109(2):72-79. 176. Arizmendi-Puga NG, Enciso JA, Ortega-Pierres G et al. Trichinella spiralis: histamine secretion induced by TSL-1 antigens from unsensitized mast cells. Exp Parasitol 2006; 114(2):67-76. ”” @   * ^ƒ} > ƒ *>   =  "    @ $      activation and survival. Infect Immun 1998; 66(6):2755-2761. 178. Leal-Berumen I, Snider DP, Barajas-Lopez C et al. Cholera toxin increases IL-6 synthesis and decreases TNF-alpha production by rat peritoneal mast cells. J Immunol 1996; 156(1):316-321. 179. Sugimoto K, Kasuga F, K Kumagai S. Effects of B subunit of cholera toxin on histamine release from rat peritoneal mast cells. Int Arch Allergy Immunol 1994; 105(2):195-197. 180. Supajatura V, Ushio H, Wada A et al. Cutting edge: VacA, a vacuolating cytotoxin of Helicobacter pylori,   !        $   {  < > $ 2002; 168(6):2603-2607. 181. Scheffer J, Konig W, Braun V et al. Comparison of four hemolysin-producing organisms (Escherichia coli,       # #  ƒ  ™    {  mediators from various cells. J Clin Microbiol 1988; 26(3):544-551. 182. Konig W, Faltin Y, Scheffer J et al. Role of cell-bound hemolysin as a pathogenicity factor for Serratia infections. Infect Immun 1987; 55(11):2554-2561. 183. Gekara NO, Westphal K, Ma B et al. The multiple mechanisms of Ca2 signalling by listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes. Cell Microbiol 2007; 9(8):2008-2021. 184. Ohkuni H, Todome Y, Watanabe Y et al. Studies of recombinant streptococcal pyrogenic exotoxin B/cysteine protease (rSPE B/SCP) in the skin of guinea pigs and the release of histamine from cultured mast cells and basophilic leukocytes. Indian J Med Res 2004; 119 Suppl:33-36. 185. Metz M, Magerl M, K Kuhl NF et al. Mast cells determine the magnitude of bacterial toxin-induced skin { ="   _••€– —˜_™“•^““ 186. Komisar J, Rivera J, Vega A et al. Effects of staphylococcal enterotoxin B on rodent mast cells. Infect Immun 1992; 60(7):2969-2975. 187. McCurdy JD, Lin TJ, Marshall JS. Toll-like receptor 4-mediated activation of murine mast cells. J Leukoc Biol 2001; 70(6):977-984. 188. Wierzbicki M, Brzezinska-Blaszczyk E. Diverse effects of bacterial cell wall components on mast cell degranulation, cysteinyl leukotriene generation and migration. Microbiol Immunol 2009; 53(12):694-703. 189. Villasenor-Cardoso MI, Salaiza N, Delgado J et al. Mast cells are activated by Leishmania mexicana LPG and regulate the disease outcome depending on the genetic background of the host. Parasite Immunol 2008. 190. Andrasfalvy M, Prechl J, Hardy T et al. Mucosal type mast cells express complement receptor type 2 (CD21). Immunol Lett 2002; 82(1-2):29-34. 191. Hartmann K, Henz BM, Kruger K -Krasagakes K S et al. C3a and C5a stimulate chemotaxis of human mast cells. Blood 1997; 89(8):2863-2870. 192. Mousli M, Hugli TE, Landry Y et al. A mechanism of action for anaphylatoxin C3a stimulation of mast cells. J Immunol 1992; 148(8):2456-2461. 193. Nilsson G, Johnell M, Hammer CH et al. C3a and C5a are chemotaxins for human mast cells and act through distinct receptors via a pertussis toxin-sensitive signal transduction pathway. J Immunol 1996; 157(4):1693-1698. 194. Fureder W, Agis H, Willheim M et al. Differential expression of complement receptors on human basophils and mast cells. Evidence for mast cell heterogeneity and CD88/C5aR R expression on skin mast cells. J Immunol 1995; 155(6):3152-3160. 195. Oskeritzian CA, Zhao W, Min HK K et al. Surface CD88 functionally distinguishes the MCTC from the MCT type of human lung mast cell. J Allergy Clin Immunol 2005; 115(6):1162-1168. 196. Soruri A, Grigat J, Kiafard Z et al. Mast cell activation is characterized by upregulation of a functional anaphylatoxin C5a receptor. BMC Immunol 2008; 9:29. 197. Ali H, Ahamed J, Hernandez-Munain C et al. Chemokine production by G protein-coupled receptor activation in a human mast cell line: roles of extracellular signal-regulated kinase and NFAT [In Process Citation]. J Immunol 2000; 165(12):7215-7223. 198. Ghebrehiwet B, Kew RR, Gruber BL et al. Murine mast cells express two types of C1q receptors that are involved in the induction of chemotaxis and chemokinesis. J Immunol 1995; 155(5):2614-2619.

184

MAST CELL BIOLOGY

199. Zutter MM, Edelson BT. The alpha2beta1 integrin: a novel collectin/C1q receptor. Immunobiology 2007; 212(4-5):343-353. 200. Barrocas AM, Cochrane DE, Carraway RE et al. Neurotensin stimulation of mast cell secretion is receptor-mediated, pertussis-toxin sensitive and requires activation of phospholipase C. Immunopharmacology 1999; 41(2):131-137. _• † ‚@ ƒ%  @#$ #  `$ <   #   $       characterization. J Immunol 1987; 138(3):861-867. 202. Bischoff SC, Schwengberg S, Lorentz A et al. Substance P and other neuropeptides do not induce mediator release in isolated human intestinal mast cells. Neurogastroenterol Motil 2004; 16(2):185-193. _•Œ @#$ #  ^ƒ  @$  >\ ~$ ^$     #$ <     # Allergy Appl Immunol 1991; 94(1-4):310-318. 204. Church MK, Lowman MA, Robinson C et al. Interaction of neuropeptides with human mast cells. Int Arch Allergy Appl Immunol 1989; 88(1-2):70-78. 205. Levi-Schaffer F, Shalit M. Differential release of histamine and prostaglandin D2 in rat peritoneal mast cells activated with peptides. Int Arch Allergy Appl Immunol 1989; 90(4):352-357. 206. Lowman MA, Benyon RC, Church MK. Characterization of neuropeptide-induced histamine release from human dispersed skin mast cells. Br J Pharmacol 1988; 95(1):121-130. 207. Alexacos N, Pang X, Boucher W et al. Neurotensin mediates rat bladder mast cell degranulation triggered by acute psychological stress. Urology 1999; 53(5):1035-1040. 208. K Kruger PG, Aas P, Onarheim J et al. Neurotensin-induced release of histamine from rat mast cells in vitro. Acta Physiol Scand 1982; 114(3):467-469. 209. Selbekk BH, Flaten O, Hanssen LE. The in vitro effect of neurotensin on human jejunal mast cells. Scand J Gastroenterol 1980; 15(4):457-460. 210. Matsushima H, Yamada N, Matsue H et al. The effects of endothelin-1 on degranulation, cytokine and growth factor production by skin-derived mast cells. Eur J Immunol 2004; 34(7):1910-1919. 211. Uchida Y, Ninomiya H, Sakamoto T et al. ET-1 released histamine from guinea pig pulmonary but not peritoneal mast cells. Biochem Biophys Res Commun 1992; 189(2):1196-1201. 212. Theoharides TC, Donelan JM, Papadopoulou N et al. Mast cells as targets of corticotropin-releasing factor and related peptides. Trends Pharmacol Sci 2004; 25(11):563-568. 213. Malaviya R, Gao Z, Thankavel K et al. The mast cell tumor necrosis factor alpha response to FimH-expressing Escherichia coli is mediated by the glycosylphosphatidylinositol- anchored molecule CD48. Proc Natl Acad Sci USA 1999; 96(14):8110-8115. 214. Mullaly SC, K Kubes P. The role of TLR2 in vivo following challenge with Staphylococcus aureus and prototypic ligands. J Immunol 2006; 177(11):8154-8163. 215. Malaviya R, Abraham SN. Role of mast cell leukotrienes in neutrophil recruitment and bacterial clearance in infectious peritonitis. J Leukoc Biol 2000; 67(6):841-846. 216. Malaviya R, Ikeda T, Abraham SN. Contribution of mast cells to bacterial clearance and their    $  "   $      =  $ ƒ _••’– 91(2-3):103-111. 217. Thakurdas SM, Melicoff E, Sansores-Garcia L et al. The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections. J Biol Chem 2007; 282(29):20809-20815. 218. Xu X, Zhang D, Lyubynska N et al. Mast cells protect mice from Mycoplasma pneumonia. Am J Respir Crit Care Med 2006; 173(2):219-225. 219. Wei OL, Hilliard A, Kalman D et al. Mast cells limit systemic bacterial dissemination but not colitis in response to Citrobacter rodentium. Infect Immun 2005; 73(4):1978-1985. 220. Ding H, Nedrud JG, Wershil B et al. Partial protection against Helicobacter pylori in the absence of mast cells in mice. Infect Immun 2009; 77(12):5543-5550. 221. Ebmeyer J, Furukawa M, Pak K et al. Role of mast cells in otitis media. J Allergy Clin Immunol 2005; 116(5):1129-1135. 222. Gekara NO, Weiss S. Mast cells initiate early anti-Listeria host defences. Cell Microbiol 2008; 10(1):225-236. 223. Ketavarapu JM, Rodriguez AR, Yu JJ et al. Mast cells inhibit intramacrophage Francisella tularensis replication via contact and secreted products including IL-4. Proc Natl Acad Sci USA 2008; 105(27):9313-9318. 224. Carlos D, Frantz FG, Souza-Junior DA et al. TLR2-dependent mast cell activation contributes to the control of Mycobacterium tuberculosis infection. Microbes Infect 2009;11(8-9):770-778. 225. Siebenhaar F, Syska W, Weller K et al. Control of Pseudomonas aeruginosa skin infections in mice is mast cell-dependent. Am J Pathol 2007; 170(6):1910-1916. 226. Echtenacher B, Mannel DN, Hultner L. Critical protective role of mast cells in a model of acute septic peritonitis [see comments]. Nature 1996; 381(6577):75-77.

THE MAST CELL IN INNATE AND ADAPTIVE IMMUNITY

185

227. Prodeus AP, Zhou X, Maurer M et al. Impaired mast cell-dependent natural immunity in complement @Œ^   ~$  €€”– Œ€•˜“““™”_^” 228. Maurer M, Lopez Kostka S, Siebenhaar F et al. Skin mast cells control T-cell-dependent host defense in Leishmania major infections. FASEB J 2006; 20(14):2460-2467. 229. Wershil BK, Theodos CM, Galli SJ et al. Mast cells augment lesion size and persistence during experimental Leishmania major infection in the mouse. J Immunol 1994; 152(9):4563-4571. _Œ• <$     `    @$$ #   ^  &&!   Immun 1993; 61(5):2242-2244. 231. Urban JF Jr, Schopf L, Morris SC et al. Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast cell- and T-cell-dependent mechanism. J Immunol 2000; 164(4):2046-2052. 232. McDermott JR, Bartram RE, K Knight PA et al. Mast cells disrupt epithelial barrier function during enteric nematode infection. Proc Natl Acad Sci USA 2003; 100(13):7761-7766. 233. K Knight PA, Wright SH, Lawrence CE et al. Delayed expulsion of the nematode Trichinella spiralis in  < # $  ^   $ # $    ^ > ="  2000; 192(12):1849-1856. 234. Crowle PK. Mucosal mast cell reconstitution and Nippostrongylus brasiliensis rejection by W/Wv mice. J Parasitol 1983; 69(1):66-69. 235. Urban JF Jr, Noben-Trauth N, Schopf L et al. Cutting edge: IL-4 receptor expression by nonbone marrow-derived cells is required to expel gastrointestinal nematode parasites. J Immunol 2001; 167(11):6078-6081. 236. Furuta T, Kikuchi T, Iwakura Y et al. Protective roles of mast cells and mast cell-derived TNF in murine malaria. J Immunol 2006; 177(5):3294-3302. 237. Abe T, Nawa Y. Worm expulsion and mucosal mast cell response induced by repetitive IL-3 administration in Strongyloides ratti-infected nude mice. Immunology 1988; 63(2):181-185. _Œ— $ ` $<$   š       ^  &&!   ‹$  resistance against larval Haemaphysalis longicornis ticks. J Parasitol 1985; 71(4):443-448. 239. Yano H, Wershil BK, Arizono N et al. Substance P-induced augmentation of cutaneous vascular permeability   $          > @ ! €—€– —’˜’™_”“^_—“

CHAPTER R 11

& $ % +

)%(*$ )&+(%k IMMUNOMODULATION Mindy Tsai,1 Michele Grimbaldeston3  # > |,1,2 1

Department of Pathology, 2Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA; 3Division of Human Immunology, Centre for Cancer Biology, and Schools of Molecular and Biomedical Sciences or Medicine, University of Adelaide, Adelaide, South Australia, Australia    3 ]    <  

Abstract:

       #    # $     %# environmental antigens, invading pathogens or environmentally-derived toxins. Mast cells also can undergo alterations in phenotype, anatomic distribution and numbers during innate or adaptive immune responses. In addition to their well-known roles as effector cells during IgE- and antigen-induced allergic reactions, mast cells can be activated by many other signals, including some that are derived directly from pathogens or which are generated during innate or adaptive immune responses. Mast cells also express many costimulatory molecules with immunoregulatory activities and can secrete many products that can positively or negatively regulate immune responses. In this chapter, we describe mouse models used for analyzing mast-cell function in vivo and illustrate how such models have been used to identify !  ! $$      $      ! $  &   {     # ^ functions, products and surface receptors that have the potential to permit mast cells to promote or suppress immune responses that can either enhance host defense or contribute to disease.

INTRODUCTION Mature mast cells are long-lived tissue resident cells distributed widely throughout vascularized tissues. Large numbers of mast cells can be found near body surfaces in the skin, airways and gastrointestinal tract.1-3 Mast cells, with dendritic cells (DCs) and  #$      #    # $    

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 186

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

187

with environmental antigens and allergens, invading pathogens or environmentally-derived toxins. Mast cells can re-enter the cell cycle and proliferate following appropriate stimulation and increased recruitment and/or retention and local maturation of mast-cell progenitors can also contribute to the expansion of mast-cell populations in tissues.1-4 Expansion of mast cell numbers, local changes in their tissue distribution and alteration in their phenotypic #    $   $    {  $ 1-4 In addition to their well-known function in IgE-dependent responses, mast cells can respond to a variety of innate signals derived from pathogens, venomous animals, or the activation of complement, or from other host cell types, such as neurons.5-9 Moreover, mast cells are equipped with a wide spectrum of costimulatory molecules that have immunoregulatory functions and also represent a potential source of many potent chemical mediators, growth factors, chemokines and cytokines, some of which can be rapidly released upon mast cell activation. With properties such as these, mast cells have the potential to regulate the transition from innate to acquired immune responses through effects that can either enhance or suppress the development, survival, proliferation, migration, maturation, or function of other immune cells.2,3,6,7,10-13 During individual biological responses, mast cells can function as effector cells, immunoregulatory cells, or both (Table 1). Mast cells can function as effector cells during innate6,8,11,14,15 or acquired6,7,10,12,13,16 immune responses. “Effector functions” of mast cells include killing pathogens,11,14,15 degrading potentially toxic endogenous peptides17-19 or components of venoms18,20 and regulating the numbers, viability, distribution, phenotype  ‰^$Š $   $$   $#     !$ # cells. Mast cells can exert effector functions through the direct or indirect actions of a wide spectrum of mast-cell-derived products and such effects can be observed in both innate6,11,14 and acquired6-8,10,12,13 immune responses.

    {$    #   $    herein as cells of hematopoietic origin that participate in innate or acquired immune responses, including granulocytes, monocytes/macrophages, DCs, T cells, B cells, NK K and NKT cells, etc. Effects of mast cells on the recruitment, survival, development, phenotype  $  $    #     ‰$$  $Š Through effector and/or immunomodulatory functions, mast cells can promote the     # $  # { $    some cases, tissue injury associated with immune responses, including innate or adaptive immune responses to pathogens as well as allergic or autoimmune disorders.6-8,10-16,21 In vitro studies suggest that mast cells also can promote the development and extent of acquired immune responses through functions such as antigen presentation and many different effects on the biology of DCs, T cells and B cells.12,15,22 However, few of these  $ #!     !! Given the many mechanisms by which mast cells can enhance the initiation or magnitude of immune responses, mast cells are often thought of as cells whose primary role is to “turn immune responses on”. However, several lines of recent evidence indicate mast cells can  $ # { $   $ ‘$   %# $ responses.12,13,23,24 Accordingly, a new picture of the mast cell is emerging: these cells have the potential to help turn immune responses off, as well as to turn them on. T cells specialized to down regulate immune responses are referred to as T regulatory (TReg) cells.25 However, #      !  # "     !  # distinct “subset” of “immunoregulatory mast cells” specialized to down- regulate or $  $    % #  %   ^{  

188

MAST CELL BIOLOGY

Table 1. Effector and immunomodulatory functions of mast cellsa Effector functionsb Promote clearance of pathogens by phagocytosis and/or secretion of antimicrobial peptides Degrade potentially toxic endogenous peptides and components of venoms Increase vascular permeability (e.g., by histamine) Stimulate bronchial smooth muscle–cell contraction (e.g., by leukotriene C4) \     # ˜   ™ Immunomodulatory functionsc Examples of positive immunomodulatory functions Promote the migration, maturation, differentiation and function of immune cells via secretion of factors such as TNF, chemokines, histamine, LTB4 and proteases Present antigen to T cells (via MHC class I or II molecules) or enhance Antigen presentation by capturing IgE-bound-antigen via Fc¡RI and then undergoing apoptosis Promote B-cell IgE production (through IL-4, IL-13 and CD40L) L Promote B-cell IgA production (through IL-6 and mast cell-B cell proximity) Promote expression of TSLP on epithelial cells (for example, by TNF, IL-4 and IL-13) Promote recruitment of immune cells by production of TNF and other mediators that upregulate adhesion molecule expression on vascular endothelial cells Promote TH2 responses via effects of prostaglandin D2 on DC maturation Promote airway smooth muscle production of chemokines and cytokines (via TNF, IL-4 and IL-13) In the presence of physiological levels of IgE, promote sensitization in certain models of CHS (via increased migration of skin DCs and perhaps other functions) In response to certain activators of mast cells used in conjunction with vaccines, enhance development of protective adaptive immune responses to pathogens Examples of negative immunomodulatory functions Suppress sensitization for CHS (via UVB-induced production of histamine) \   #     <   ˜! # #     ™ Mediate Anopheles mosquito-bite-induced suppression of development of certain *^^  ˜!     #     ™ Suppress many features of certain models of severe CHS, in part via effects of IgG1/mast cell-FcaR-dependent production of IL-10 Mediate jet fuel-induced suppression of CHS (via PGE2 and perhaps IL-10) Suppress many features of the pathology of chronic low-dose UVB irradiation of the skin, in part via effects of 1_,25-dihydoxyvitamin D3-mast cell-VD - R-dependent production of IL-10 continued on next page

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

189

Table 1. Continued Suppress cytokine production by T cells and monocytes (via IL-10) $   $   ^{  <  #<  <  (via IL-10) Enhance ability of DCs to reduce T-cell proliferation and cytokine production (via IL-10) a) *#        >   ~ ‚! $ _••—– —’”—^—“ ˜  Œ™ %#   from Macmillan Publishers Ltd. Please see the original source for relevant references. b) Effector functions include the non-immunomodulatory physiological or pathological functions of mast cells or the direct regulation of ‘‘nonimmune’’ cells, such as vascular endothelial cells, epithelial     !   $  c) These are effects on other immune cells (such as DCs, T cells, B cells, monocytes/macrophages and granulocytes) and effects on structural cells (such as vascular endothelial cells, epithelial cells and # $ ™ #  #   {$ $ 

immunosuppressive functions of mast cells as “negative immunomodulatory” functions and to those functions that enhance the initiation, magnitude or duration of immune responses as “positive immunomodulatory” functions” (Table 1). In this chapter, we highlight mouse models used for analyzing mast-cell function in vivo and illustrate how such models have been utilized to identify immunomodulatory     $    $  &   {    of the mast-cell functions, products and surface receptors that have the potential to contribute to the mast cell’s ability to promote or suppress immune responses (reviewed in refs. 6,7,9-11,13,16).

MOUSE MODELS OF MAST-CELL FUNCTION

@ \>@+  #$#  #   <    #!     -kitt mutant  %##        $ #! !  # #     available for analyzing the in vivo functions of mast cells.1,6,26 The most commonly used animals for such studies are the WBB6F1-KitW/W-vv mice and C57BL/6-KitW-sh/W-sh mice.6,7,26-28 KitW is a point mutation that produces a truncated Kit, lacking the transmembrane domain, that is not expressed on the cell surface; KitW-v is a (Thr660AMet) mutation at the c-kit tyrosine kinase domain that substantially reduces the kinase activity of the receptor; and KitW-sh is an inversion mutation that affects the transcriptional regulatory elements upstream of the c-kitt transcription start site on mouse chromosome 5 (reviewed in refs. 6,7,28). Adult WBB6F 1-KitW/W-v mice and C57BL/6-KitW-sh/W-sh mice are profoundly       1,6,26,27 WBB6F1-KitW/W-v mice exhibit several other phenotypic abnormalities, such as macrocytic anaemia, reductions in numbers of bone-marrow and blood neutrophils, sterility and an almost complete loss of interstitial cells of Cajal.1,6,26 By contrast, C57BL/6-KitW-sh/W-sh mice are neither anaemic nor sterile, but have increased numbers of bone-marrow and blood neutrophils, enlarged spleens and mild cardiomegaly.26,28-30

190

MAST CELL BIOLOGY

Because the c-kit-related phenotypic abnormalities that affect lineages other than mast cells are generally milder in C57BL/6-KitW-sh/W-sh mice than in WBB6F1-KitW/W-v mice and because C57BL/6-KitW-sh/W-sh mice are fertile and, like many other mutant mice to which they might be bred, are fully on the C57BL/6 background, they are becoming increasingly popular for studies to elucidate the roles of mast cells in vivo. Herein, we will refer to both WBB6F1-KitW/W-v and C57BL/6-KitW-sh/W-sh mice as “c-kitt mutant mice”. Differences in the biological responses in c-kitt mutant mice compared with wild-type mice may be due to any one of the genetic abnormalities in these animals and may not be due to the loss of mast cells. However, the lack of mast cells in c-kitt mutant mice can be selectively repaired by the adoptive transfer of genetically-compatible, in-vitro-derived wild-type or mutant mast cells.1,6,26,27 Such in vitro-derived mast cells, for example bone-marrow-derived cultured mast cells (BMCMCs), can be administrated to c-kit mutant mice intravenously, intraperitoneally or intradermally, or directly injected into the anterior wall of the stomach, to create so-called “mast-cell knock-in mice”. These mast-cell knock-in mice can then be used to assess the extent to which differences from wild-type mice in the expression of biological responses observed in c-kitt mutant ^^   { # <    &  ! k    @   *#     ^^     !  !!  testing animals in which that mediator has been knocked out. To the extent that the   ! "             {$ # "   #    $ #    % $ about the role of that mast cell mediator in vivo. For example, in mice that lack mouse mast-cell carboxypeptidase A (mMC-CPA, also known as mast cell-CPA and CPA3; a highly conserved secretory granule protease), the expression of mouse mast-cell protease 5 (mMCP-5) is also reduced because it requires mMC-CPA for proper packaging in the cytoplasmic granules.31 This problem can be circumvented by using mice in which  @^@\ #  $       !  # # preserves mMC-CPA’s ability to ensure proper packaging of mMCP-5 in the mast cell granule.20 Mice that lack mast cell protease-1 (MCPT-1),32 MCPT-4,33 tryptase beta 2 (TPSB2; also known as MCPT-6),34 or mast cell-CPA/CPA3,31 or that have a mutated form of mast cell-CPA/CPA3 that essentially lacks enzymatic activity,20 have been used  } %## #   #   ˜ # } !™ {$ other aspects of mast-cell phenotype, such as content of other stored mediators, and to   # $  $#    !! *   "  @ ^  $ #    ‰   Š promoters recently have been generated.35-37 Such “mast cell cre mice” are being crossed %# #     %## #       ‰{"Š    $ # "       $  ˜     ™  #   lineage. Such approaches may prove to be useful in attempts to analyze to what extent mast cells represent important sources of products (including those with potential effector and/or immunomodulatory functions) that can also be derived from other cell types. “Mast cell cre” mice could also be mated to other transgenic mice in which important mast cell $ !!     {"        ! *#  #    #    ‰ !Š  ^  $  #    of c-kitt mutations. However, time will tell whether various “mast cell cre” mice achieve

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

191

 $ ^^  "   @   !    $   all mast cell populations without affecting other cell lineages. (  Pharmacological approaches or those based on the use of antibodies to deplete mast cells or to neutralize their products may also provide useful information, but are limited  #    #  $   #   $#  # block the effects of that mediator whether it is secreted from mast cells or other cells. Antibodies that neutralize SCF38 or block Kit39-41 can result in the depletion of mast cells  !! $   {$ #   # "   Drugs (or antibodies) that only interfere with mast-cell activation would be highly desirable for experimental studies and, possibly, for evaluation as therapeutic agents. One drug, disodium cromoglycate, is widely characterized as a “mast-cell stabilizer” (i.e., an agent that blocks the release of mast cell mediators that occurs upon appropriate activation of the cell) and sometimes is used to suppress mouse mast-cell function in vivo,42,43 $  $      $   `%!  #      restricted to mast cells44  #  $  {$  $  †^ $45 Given the current limitations of using pharmacological or antibody-based approaches         < # $ ! % #< #   # $ #    <<^        ^^    %# # #!  $ !  # #       !      #   ! %    #  }   $  !!

MAST CELL ACTIVATION Mast cells can be activated by a wide range of stimuli, including those that activate immunoglobulin Fc receptors (Fc¡RI, FcaR), complement receptors (C3aR, C5aR) and microbial pattern-recognition receptors (PRRs, such as TLRs). Mast cells also respond to many other signals, including neuropeptides, cytokines, growth factors, toxins, venoms or venom components and physical stimuli. These stimuli trigger mast cells to release a diverse array of biologically active products, many of which can potentially mediate  ^{  ^{   $$ ! $13 The strength and nature of the responsiveness of mast cells to various activating stimuli may be {$      !    #  # "    or functional properties of the surface receptors or signaling molecules that contribute to such responses.6,46-48 Furthermore, mast cells can participate in multiple cycles of activation for mediator release and can be differentially activated to release distinct patterns of mediators or cytokines, depending on the type and strength of the activating stimuli.6,49 Activation via Fc¡*+  ( ^ * Mast cells and basophils are the two major cell populations in the mouse that $! "   # $    $   # ##     = Fc¡RI and the number of surface Fc¡RI is up-regulated by increased concentrations of IgE.3 Aggregation of Fc¡RI by binding of bi- or multi-valent antigen to surface- Fc¡RI

192

MAST CELL BIOLOGY

-bound IgE on mast cells initiates complex intracellular biochemical events that lead to degranulation and secretion of mast-cell-derived products and mediators. This Fc¡RI-dependent mast cell activation response results in rapid release (in minutes) of preformed cytoplasmic granule-associated mediators (such as histamine, heparin and other proteoglycans, proteases) and certain cytokines (TNF, VEGF), the secretion of de novo-synthesized lipid mediators (including cysteinyl leukotrienes [LTs] and prostaglandins [PGs]) and the production, with a prolonged kinetics, of many cytokines, chemokines and growth factors.3,6,50 Aggregation of only a small fraction of the mast cell’s Fc¡‚  $     mast cell activation and mediator secretion; as a result, individual mast cells can be $$ }         3 As discussed above, the extent to which mast cells secrete various types of mediators can vary according to the strength of the activation signal, with release of some cytokines occurring at lower antigen concentrations than the concentration required to induce substantial degranulation and release of stored mediators.49 For example, low occupancy or weak stimulation of Fc¡RI can induce mast cells to produce “pro-allergic” chemokines, while mast cell IL-10 production requires strong and prolonged stimulation of Fc¡RI.51 The extent of mast cell activation in response to Fc¡RI stimulation can also be positively or negatively regulated by the cells’ exposure to ligands for many other receptors.6,49,50 Antigen- and IgE-dependent mast cell activation is widely regarded to be a, if not the, major initiator of the clinical signs and symptoms of an allergic reaction and can also contribute to later consequences of allergen exposure, by promoting local {        #     $  In addition to Fc¡RI, mast cell activation can also be triggered by Fca receptors. IL-4 primed-mouse mast cells can produce TNF, IL-10 and VEGF upon IgG1/FcaRIII dependent activation24,52,53 and aggregation of FcaRI on IFNa-treated human mast cells can induce histamine release.54 &*  ( + *@  "

  !      {$ # !  ‹$  immune responses.7,13,55-62 Mast cells express many Pattern Recognition Receptors (PRRs).63-65 Protein or mRN R A of 10 TLRs (TLRs 1–10) have been detected in human or mouse mast cells.65 Mast cell responses to TLR R agonists can vary among different populations of mast cells. For example, the stimulation of TLR4 on mouse mast cells by LPS resulted in cytokine production without degranulation, while TLR2 activation led to both cytokine release and degranulation.66 Mast cells activated by TLR3 stimulation (e.g., with Poly(I:C) or Newcastle disease virus) produced antiviral cytokines (IFN-`, ISG15) and chemokines (IP10, RAN A TES) without undergoing degranulation.67 Human and mouse Mast cells express NLRs (the second major class of cytoplasmic innate immune sensors), such as NOD receptors and NLRP proteins,68-71 which can be activated by intracellular pathogens and “danger signals”.72-74 Expression of various PRRs thus permits mast cells to respond to both Pathogen-Associated Molecular Patterns (PAMPs) and “danger signals” resulting from cell stress or injury. Moreover, studies in mice indicate that activation of   ! # ~ƒ‚\Œ {   $  ƒ^` overproduction and chronic urticarial rash in subjects with cryopyrin-associated periodic syndrome, a disorder associated with NLRP3 mutations.71

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

193

In conclusion, mast cell secretion of cytokines and other mediators in response to nonFcR-dependent innate stimuli, such as to TLR R agonists, complement products and   { !  # #   {$ # # $  and function of Antigen Presenting Cells (APCs) and other immune cells and thus to modulate the subsequent T-cell or B-cell responses.6-8,12

IMMUNOMODULATORY EFFECTS ON DENDRITIC CELLS Both in vitro and in vivo evidence indicate that mast cells have the potential to {$ @   $   $6,7,13 Histamine, abundantly stored in mast cell granules and rapidly released by the process of mast cell degranulation, can induce chemotaxis,75 alter the pattern of secreted cytokines76-78 and enhance expression of costimulatory and MHC class II molecules in DCs;77 and histamine-conditioned DCs have been shown to preferentially induce TH2 polarization.76,77 In vitro coculture of IgE antigen-activated umbilical cord blood-derived human mast cells with human monocyte-derived DCs induced maturation of DCs, but potently suppressed IL-12p70 production by DCs and thus promoted their TH2 polarization.79 Other mast cell products, such as PGD280 or PGE2,81 have also been shown to inhibit IL-12 production by DCs and to induce maturation of DCs toward an effector DC2 phenotype, again leading to the polarization of naive T cells to TH2 cells. These in vitro observations are in accord with in !!  #% #  $}  ‘$ %# #     # $    $ $     *H1 responses while enhancing TH2 responses.82 Many other mast cell products, such as PGD2,80,83,84 LTB4;85 and PGE2, TNF, IL-1, IL-16, IL-18 and CCL5 (reviewed in ref. 7), have also been shown to modulate DC migration, differentiation, maturation and function. Another mechanism by which mast cells can regulate DC function is via exosomes, small vesicles implicated in the transfer of materials among cells. Mecheri and his colleagues have shown that endocytosed antigens are accumulated in association with heat shock protein hsp60 and hsp70 in mast cell exosomes. Such mast cell-derived exosomes are highly potent in inducing maturation (CD40 and CD80 expression) and $ ! ˜   ƒ^_”•™  @    #   of antigen presentation by DCs.86 ‚! % $ #! ! #      {$ @ biology in vivo. In a model of contact hypersensitivity (CHS) to oxazolone, Bryce et al reported evidence that the emigration of skin Langerhans Cells (LCs) from the epidermis    $$   "} %    ^^  WBB6F1-Kit - W/W-vv mice and was enhanced by antigen-independent effects of IgE.87 Moreover, this group showed that mice lacking IgE exhibited impaired elevation of mRN R A for the mast-cell-associated product, MMCP-6 (also known as MCPT-6 or TPSB2), as well as for several products (that can be produced by mast cells and other cell types) known to {$ @  ˜$#  ƒ^ ƒ^“ @@ƒ_  *~™  #$  # $$ application of the hapten oxazolone.87 These results suggest that the binding of IgE to Fc¡RI on dermal mast cells, even in the absence of antigen known to be recognized by that IgE, in some way can “prime” such mast cells to participate more effectively in the induction of DC migration and perhaps other functions that promote sensitization to the hapten.87 Histamine derived from IgE- and antigen-stimulated mast cells in mouse skin can promote the H2 receptor-dependent migration of LCs to draining lymph nodes (LNs)88

194

MAST CELL BIOLOGY

 *~  !   $ ^   $    #    FITC-induced migration of cutaneous and airway DCs.89 Administration of peptidoglycan can result in mouse mast cell-dependent L LN hypertrophy, LC mobilization90 and recruitment

of plasmacytoid and CD8 DCs to draining LNs.91 While peptidoglycan-induced LN hypertrophy was TNF-independent in this model, optimal LC migration required TNF.90 Activation of dermal mast cells by a TLR7 agonist also has been shown to promote the emigration of LCs, via a process partially dependent on mast-cell-derived IL-1.56

IMMUNOMODULATORY EFFECTS ON LYMPHOCYTES Evidence for the migration of mast cells to draining LNs during immune responses  #   "     #    $ {6,12,92,93 $ #    #  {$ # # § $  bidirectional cell–cell interactions. Regulation of Mast Cell Functions by T Cells Upregulation of IL-8 mRN R A transcription has been demonstrated in mast cells that were in contact with the membrane of activated T cells.94 Incubation of mast cells with activated lymphocytes induced mast cell degranulation and release of granule-associated mediators/proteases (histamine, `-hexosaminidase, metalloproteinase-9 and tissue inhibitor of metalloproteinase [TIMP]1), TNF and other cytokines.95-98 ICAM-1/LFA-1 dependent cell-cell contact is necessary for anti-CD3-activated T cells to augment Fc¡RI-dependent mast cell (BMCMC) degranulation and histamine release96 and the interactions between LT_1`2 and/or LIGHT on activated T cells and LT` receptors expressed on mast cells are important for mast cell cytokine release induced on contact with activated T cells.98 Mast Cell Effects on T-Cell Functions Mast cells can also modulate T-cell functions. Antigen processing and presentation has been proposed as one mechanism by which mast cells might regulate T-cell responses.7,92,99 Mecheri and his colleagues demonstrated upregulation of MHC class II in BMCMCs upon stimulation with LPS and reported that BMCMCs can take up, process and present antigen      * #  22 Rat peritoneal mast cells (PMCs)100 and human mast cells101 were also later reported to be capable of antigen presentation. MHC class I molecules are expressed on mouse BMCMCs and on human mast cells isolated from lung, liver, uterus and skin99 and, in vitro, mouse BMCMCs can stimulate bacterial antigen<  @— *^ !       # $# `@ class I.102 Recently, Stelekati et al showed that BMCMCs can present antigen to CD8 T cells in a MHC I-restricted manner, resulting in IL-2, IFNa and MIP-1_ production and promoting CD8 T-cell degranulation and cytotoxicity.103 #$#    than professional APCs, MHC II-expressing mast cells can upregulate CD69 expression, proliferation and cytokine production in effector T cells.104,105 The expression of MHC molecules and antigen presenting functions in mast cells appear to be under complex control: MHC II expression is down-regulated by IL-3, but upregulated by IL-4 and IFN-a.101,104,106 ^@ %#  {$ "  of MHC II, can increase CD80 and CD86107 expression and substantially enhance the

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

195

antigen-presenting capacity of IL-4-treated mast cells. By contrast, IFN Na almost completely abolished mast cell antigen-presenting function.106 In another study, Delta-like 1 (Dll1)/ Notch signaling was shown to induce the expression of MHC-II and upregulate the expression of OX40L on mast cells, thus promoting the ability of MHCII OX40Lhigh BMCMCs to enhance naïve T-cell proliferation and their differentiation into IL-4, IL-5, IL-10 and IL-13 secreting TH2 cells.108 Treatment of cultured mast cells (BMCMCs and spleen-derived mast cells) with LPS/IFNa also enhanced mast cell expression of MHC-II and the inhibitory costimulatory molecule PD-L1, but not positive costimulatory B7 family members CD80 and CD86 and promoted the mast cells’ ability to process and present antigen directly to previously activated effector CD4 T cells and, to a lesser degree, to naïve T cells. Furthermore, LPS/IFNa-primed mast cells stimulated expansion  ^  "Œ TReg cells preferentially over naïve T cells.105 *#  may have implications for understanding the mechanisms by which mast cells can exert ^{  $    *^  ‹$  24,109 Although MHC class II molecules are not expressed on most “resting” mouse or human mast cells, expression is upregulated in mast cells that have been isolated from pathogen-infected tissues and/or stimulated by tumor necrosis factor (TNF), IFNa or bacterial lipopolysaccharide (LPS).99,105 Kambayashi et al showed that s.c. injections of LPS increase mast cell numbers in the draining LN and that these mast cells expressed MHC II, PD-L1, CD80 and CD86.105 Freshly isolated peritoneal mast cells and IFNa/ IL-4-primed mast cells, derived from peritoneal cell culture, have been shown to process protein antigen and to form functional immunological synapse with and induce activation of, effector CD4 helper T cells, but not naïve T cells.104 The cognate interactions at the immunological synapse between T helper cells and antigen-presenting mast cells and the formation of T-cell polarization at the contact of T cells and mast cells has been visualized by confocal laser scanning microscopy.104 Such direct contacts of mast cells and T helper cells also render mast cells more susceptible to Fc¡RI-dependent degranulation.104

    {$ *^ !  #   " =" derived from IL-4-treated mouse BMCMCs induce lymphocyte proliferation and IL-2 and IFNa production in vitro. Injection of such mast-cell<derived exosomes, which contain costimulatory molecules (such as MHC II, CD86, CD40, CD40L, LFA-1 and intercellular adhesion molecule 1), can induce lymphocyte proliferation and cytokine production in vivo.110 Mast cells can enhance antigen presentation indirectly in vitro by internalizing antigen bound to Fc¡RI-associated IgE; this mechanism is independent of mast-cell MHC class II expression, but requires that such mast cells undergo apoptosis and then are phagocytised by other antigen-presenting cells.111 Targeting antigens to IgE or IgG bound to mast cells  # #      112,113 which might be mediated by the mechanism described by Kambayashi et al,111 or by the transfer of antigen to other APCs via mast-cell-associated exosomes. As noted above, mouse and/or human mast cells can express many costimulatory molecules including members of the B7 family—inducible T-cell costimulator ligand (ICOSL), PD-L1, PD-L2, CD80 (also known as B7.1) and CD86 (also known as B7.2)—members of TNF–TNF receptor families—OX40, CD153, CD95, 4-1BB and glucocorticoid-induced TNF-receptor-related protein (GITR)—and CD28 and CD40 ligand (CD40L).12,114 Moreover, mast cells can exhibit costimulatory function in vitro. For example, engagement of the costimulatory ligand OX40L expressed by human114 or mouse115

196

MAST CELL BIOLOGY

mast cells and OX40 expressed by T cells is required for optimal mast-cell-dependent enhancement of T-cell proliferation114 or cytokine production.115

    $     {$ *^ !  " histamine, LTB4, PGD2 and TNF can promote the migration, recruitment, maturation and activation of lymphocytes; and PGD2 can enhance cytokine production by TH2 cells.116 Histamine promotes TH1-cell activation through H1 receptors but conversely can suppress both TH1 and TH2 cell activation through H2 receptors.117 Mast cells are also sources of many cytokines, such as IL-2, IL-4, IL-6, IL-10, IL-12, IL-13 and transforming growth factor-` (TGF-`™ #  {$ #  }  ´! *  %  *H1, TH2, TH17 and TReg cells12 and can modulate the function of distinct T-cell subsets. Taken together, mast cells can potentially modulate T-cell-dependent acquired $      *    #     presentation by other APCs. Mast cells can enhance T-cell function by direct cell-cell contact, by exosome-mediated effects and/or by their secreted products. Although there is not yet clear evidence that native populations of mast cells can perform these functions in vivo, migration of mast cells to draining LN has been shown in many innate and acquired immune responses, including CHS,118 UVB irradiation,119 EAE,120 cutaneous infection with >  =,105 injection of LPS105 and in a model of anti-GBM (glomerular basement membrane) induced glomerulonephritis.121 These observations suggest that    !      # #! #   {$ *^ function within LNs, as well as at the sites in which such mast cells ordinarily reside. Effects on CD8 T Cells !   $ #!   " # $  @— *^ function by mast cells. Mouse BMCMCs can stimulate bacterial antigen<  @— T-cell activation by presenting bacterial antigens through MHC I.102 A recent study by <     # † @ @      @— *    `@ I-restricted manner, resulting in IL-2, IFNa and MIP-1_ production and can promote CD8 T-cell degranulation and cytotoxicity.103 Intraperitoneal injection of Poly(I:C), a TLR3 ligand, induces CD8 T-cell recruitment and promotes the expression of MHC II, CD80, CD28 and complement receptors by PMC.67 `   $ # ^^  KitW-sh/W-sh  "#    ^  " *^#  after transcutaneous peptide immunization together with TLR7 ligands (used as adjuvants) and that, in mice containing mast cells, treatment with ligands for TLR7 induced mast cell IL-1-dependent LC emigration and mast cell TNF-dependent LN hypertrophy.56 Effects on B-Cells and Immunoglobulin (Ig) Production Several mast cell products, including IL-4, IL-5, IL-6, IL-13, CD40L and rat mast-cell     {$ †^ !  $ $ =  $122,123 Using a coculture system, Merluzzi and colleagues recently showed that BMCMCs can promote the survival and activation of naïve B cells and promote the proliferation and differentiation of activated B cells toward IgA secreting plasma cells.124 These effects of mast cells require both cell-cell contact and mast-cell-derived IL-6.124 Mast-cell-derived "    @ $   \@        production in naive mice in the absence of conventional adjuvants.86 In a mouse model of urinary tract infection with uropathogenic E. coli in WT mice, KitW-sh/W-sh mice and

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

197

KitW-sh/W-sh mice engrafted with WT or TNF
IMMUNOMODULATORY FUNCTIONS IN VIVO Some of the immunomodulatory functions of mast cells that have been proposed based on in vitro $ #!    in vivo using mast-cell knock-in mice or  <   ^^    <     } activity. In many of these studies, the end points assessed included the recruitment of particular immune cells, such as granulocytes, DCs or various subpopulations of #   # $  #% # # <        mast-cell product, decreased the pathology associated with the immune response or impaired its effectiveness in promoting host resistance to infection (reviewed in ref.13). &#   # $ $  #  ^{  $    several recent studies provided compelling evidence that mast cells can also down-regulate {      $ #    "   Host Defense against Infections Work by many groups, using both mast cell knock-in and mast-cell-associated  ^   # #% #    $ #   survival during several examples of bacterial infections.8,11,19,30,34,59,125-128 *#        #   $    %    by two seminal studies in 1996.125,126 In the cecal ligation and puncture (CLP) model, which is considered by some investigators to be the “gold standard” mouse model of sepsis (reviewed in refs. 17,19,30,125), host resistance and survival can be enhanced by mast cells and TNF. Treatment with SCF, that increases mast cell numbers, can provide further protection in the CLP model.129 Activation of mast cells mediated by TLR4,66 complement receptors,130,131 or endothelin-1 (see ref. 17) has been shown to contribute to the ability of mast cells to enhance host resistance in CLP models. On the other hand, Smad3 and IL-15 expression in mast cells function to inhibit mast cell-mediated protection against bacterial infection.132,133 There is evidence that MCPT-2 can contribute to neutrophil recruitment and host survival during CLP, but that, in wild-type mice, mast-cell production of intracellular IL-15 limits the mast cell’s ability to produce this protease in that setting.133 ^^  KitW/Wv mice engrafted with Smad3
198

MAST CELL BIOLOGY

phagocytosis-dependent bactericidal activities (reviewed in refs. 8,59,135,136) and proteolytic degradation of endogenous mediators which would otherwise be elevated to toxic levels, such as endothelin-1 (see ref. 17) and neurotensin.19 Secretion of proteases/ enzymes and an antimicrobial peptide137 and formation of extracellular traps that contain antimicrobial peptides, histone, DNA and tryptase,138 have also been proposed as potential mechanisms by which mast cells exert protective functions. For example, mast-cell-derived proteases can promote host defense following intraperitoneal injection of the bacteria Klebsiella pneumoniae  *\†_^   #! #   $ #  $  #   !       34 Mast cells can also contribute to host defense by modulating acquired immunity to pathogens. They can process bacterial antigen and present it to T cells,102 recruit lymphocytes and induce LN hyperplasia by TNF production.139 Mast cells and mast cell-TNF have been #%   $ *  @  # {    { $  # draining LN after E. coli infection62 and mosquito bites.140 K Kunder et al have reported that one mechanism by which mast cells regulate lymphocyte recruitment is by the delivery   {     " # ^  granules that also contain other mast cell mediators, particularly TNF, to draining LNs via lymphatic vessels.60 Such observations support the conclusion that mast cells can have important sentinel and effector roles during bacterial infection, which help to promote clearance of the bacteria, protect the host from pathology and enhance survival. However, there is evidence from work in mast-cell-engrafted C57BL/6-KitW-sh/W-sh mice that mast-cell-dipeptidyl peptidase-I can have effects in a severe model of CLP that decrease levels of IL-6 and reduce survival.141 Moreover, another study in mast-cell-engrafted C57BL/6-KitW-sh/W-sh mice indicates that in certain severe bacterial infections, including a model of severe CLP, mast cell production of TNF (and perhaps other mast cell functions) can exacerbate {   30 Another setting in which mast cell responses to bacteria may contribute to pathology is in atopic dermatitis, an allergic disorder in which the majority of patients have colonization of the skin with Staphylococcus aureus, a source of peptidoglycan which could mediate TLR2-dependent activation of mast cell cytokine production.59,142 Considerations such as these suggest that, depending on the setting, including the severity and/or type of infection or the presence of another disorder, mast cells can either promote health or increase pathology during host responses to bacteria. Intrinsic properties of the mast cell, such as genetic predispositions to produce larger or smaller amounts of TNF and other cytokines, or the presence of other abnormalities in the host (e.g., C57BL/6-KitW-sh/W-sh mice have increased numbers of neutrophils),28-30   {$ %## #       $         # $ In addition to their role in bacterial infections, mast cells can promote host resistance to certain parasite infections. However, the mechanisms involved have not been fully      " !! #    ^^ effects (reviewed in refs. 6,8,143,144™  "     *\†_ ˜  =™ was associated with markedly reduced recruitment of eosinophils to the sites of larvae deposition in skeletal muscle during the chronic phase of Trichinella spiralis infection, but was not associated with a detectable abnormality in the intestinal expulsion of the parasite.144 In a model of cutaneous parasite infection by >  =, mast cells  $  #    <     ^  *   # #  $   ^{  $ #  #  @  

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

199

   !  #    $   # $   protection in this model.143 There is evidence that mast cells participate in host responses to certain viruses, but their precise roles in such settings are not yet clear.8,145 Rodent11,59,67,134 and human11,63,146 mast cells express TLRs (e.g., TLR3) which can be activated by viral double-stranded R A to release various chemokines and cytokines, including interferon-_ and –`.63 RN Some of these mast-cell-derived products may contribute to host defense against viruses.8,11,63,67 Co-stimulation of rodent and human mast cell populations in vitro via the Fc¡‚    *ƒ‚  # # §    ! $  ^{  mediators, suggesting one mechanism whereby bacterial or viral infections might exacerbate allergic diseases and other IgE- and mast-cell-associated disorders in vivo.59,147,148 Innate Responses to UVB Irradiation Mast cells have been implicated in ultraviolet (UV)-B-induced immunosuppression and many mast-cell-associated mediators (histamine, PGE2, serotonin, PAF, TNF, IL-4, IL-10) are produced in response to UVB irradiation (wavelengths: 280-320 nm) of the skin.119,149      %  $  'ž†^$ { =*^    $$'ž† !<{ following a single exposure to UVB.150 †       ^{    model of chronic low-dose UVB-irradiation,24,151 at least in part by their ability to produce ƒ^•   # # ^{  <      "$   'ž†    $   # {    $ # including numbers of granulocytes, macrophages and T cells at the reaction sites, as well as the local tissue swelling, epidermal hyperplasia and epidermal necrosis.24,151 Hart et al23 showed that the ability of a single high dose of UVB irradiation of the skin to induce systemic immunosuppression of contact hypersensitivity (CHS) responses to the hapten 2,4,6-trinitrochlorobenzene was markedly reduced in (C57BL/6 x DBA/2) F1-KitW-f/W-ff mice but was restored following mast-cell engraftment. Several lines of evidence suggested that histamine was a major mediator of this UVB-induced, mast-cell-dependent effect.149   %       #  # 'ž†   suppressed delayed-type hypersensitivity (DTH) responses to allogeneic spleen cells in KitW-f// mice (which contain dermal mast cells) but not in KitW-f/W-ff mice (which do not contain dermal mast cells). Alard et al showed that mast cells, probably via the secretion of TNF, are required for local UVB-induced immune suppression.152 CXCR4 dependent migration of mast cells to the LN upon UVB treatment is critical for the UVB-induced immune suppression.119 More recently, Biggs et al provided evidence that activation of mast cells through their vitamin D receptors (VDRs) by physiologically active vitamin D3 (i.e., 1_,25-dihydoxyvitamin D3) is required for optimal release of mast-cell-derived ƒ^• %##  $   $  #  §   $  {  skin pathology at sites of chronic low-dose UVB irradiation (Fig. 1).151 T-Cell-Dependent Responses EAE In the mouse model of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG), mast cells can increase the incidence

200

MAST CELL BIOLOGY

Figure 1. Mast cells and mast cell VDR expression are required to limit pathology induced by chronic low-dose UVB irradiation of ear skin. Cross-sections of ears obtained from WBB6F1-Kit / (wild-type) mice (A-C), WBB6F1-KitW/W-v (KitW/W-v) mice (D-F), WT BMCMCAKitW/W-v mice (G-I), or VDR–/– BMCMCAKittW/W-v  ˜>^ƒ™  _’ #  #  "$   _ <>2 UVB (15 exposures, 2 d apart; B,C,E,F,H,I,K,L); sections stained with haematoxylin and eosin (A,B,D,E,G,H,J,K), or toluidine blue (C,F,I,L). Mice that did not receive UVB irradiation were also killed for analysis of skin histology at #   # "  ˜ >™ @|   $^#  %  #<    (D) or epidermis (Ep). Red arrowheads indicate mast cells (C,F,I,L). Scale bars in A  100 +m (or 1000 +m in inset) (for A,B,D,E,G,H,J,L inset), or 10 +m (in C,I insets). Results are representative of those obtained in the 3 different experiments performed, each with 3-4 mice analyzed per group. Reproduced with permission from Biggs L et al. J Exp Med 2010; doi:10.1084/jem.20091725.

and severity of the disorder.103,153,154 Remarkably, it appears that mast cells do not have to be within the CNS to exert at least some of their important effects in MOG-induced == *#   #$#     ^^  KitW/W-v mice with

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

201

in vitro-derived BMCMCs does not result in the appearance of mast cells in the CNS of these mice, they exhibit a CNS disease severity that is similar to that in wild-type mice. *% # #!     " #% " ^@~    {$ autoimmune responses to MOG. One mechanism is through the production of IL-4 in the LNs which enhances the development of encephalogenic TH1 cells.155 Mast cell antigen   # # #    ˆ   @— *  #   shown to also contribute importantly to EAE pathogenesis.103 More recently, Brown’s group reported that, in this model of EAE, mast cells in the meninges of the brain can # ^ ^      {       @~ including the recruitment of neutrophils by mast cell-derived TNF.156 Mouse Models of Asthma Various mouse models have been used to elucidate the roles of mast cells in allergic  % {   #     } #  $  mast cells is most obvious when the mice are sensitized and/or challenged with low doses of antigens, with adjuvant-free antigen, or sensitized with antigen by inhalation route (reviewed in refs. 157,158). In mouse models of asthma in which mast cells enhance the response, mast cells158-163  ^^ ! *~    $  #  % # !   % {159,158,160-163   #   *~   {   to promote T-cell recruitment and TH2-type cytokine production.161 Another mechanism which may contribute to “mast-cell-dependent” effects on T cells during models of allergic {  ¡RI-dependent mast cell secretion of LTB4, with subsequent recruitment   @— *  @’ *      % {164-166 Mast cells also contribute to a model of TH”^^ $ #^ $ {  !$ ˜ˆž™^# ˆž^  *^    167

 !    $   #    {  #  %  cells are required for the full development of several features of tissue remodeling, including increased numbers of mucus-producing goblet cells in the airway epithelium and increased lung collagen deposition, changes accompanied by a mast-cell-dependent exacerbation of airway hyperreactivity to methacholine.158 In this mouse model of chronic asthma, mast cell activation mediated by antibody dependent- and independent-mechanisms contributes to the full manifestation of the airway disease.158 Finally, in one model of   {  #  %  < #   # @\*^’ compared to the corresponding wild-type mice, exhibited more substantial increases in  % # !  ##    % {  #< of bronchial smooth muscle.168 *#   #       $  #   # #  %#   { The Two Faces of Mast Cells in CHS Under some experimental conditions, mast cells are required for the optimal elicitation  # {  %# $   #^$ @` ˜ !% in ref. 55). Mast cells and IgE can contribute to LC emigration and promote effective sensitization to the chemical hapten oxazolone,87 as well as contribute to the effector phase  #  $ #   $$  !  169 However, mast cells can also have negative immunomodulatory functions in some models of CHS. Mast cells markedly limited the magnitude and duration as well as promoted the resolution, of

202

MAST CELL BIOLOGY

@`  $    # # _’^ ^^{$ }™ ˜~†™  by urushiol, which is the hapten-containing sap of poison ivy or poison oak.24 In these responses, mast cells substantially inhibited the development of pathology, including the $<      #        %# # responses. Several lines of evidence indicated that mast cell production of IL-10, mediated       ^      aRIII on mast cells, is essential for   ^{  $  # 24 Although the pathways that link mast-cell-derived IL-10 (or other mast cell mediators that are relevant in this setting) to #  ! $ #         ^^ ! ƒ^•  {$ #  # $#  "        effector and immunoregulatory functions. In another study, the nature of the mast cells contribution to Ox-induced CHS was found to depend on the dose of hapten used to immunize the mice. Norman et al showed # ^^  KitW/W-vv mice, when compared to wild-type mice, exhibited reduced CHS responses (associated with little or no local production of IL-10) when immunized with low doses of Ox but more severe reactions (associated with local production of IL-10) when immunized with high doses of Ox. This study suggests that, depending on the strength of immunization, mast cells can regulate the magnitude of the T-cell-associated CHS responses by altering the cytokine microenvironment.170 Antibody-Dependent Responses Mast cells can contribute substantially to the disease pathology induced by auto-antibodies. In a mouse model of anti-glucose-6-phosphate isomerase (GPI)-induced destructive arthritis, work in KitW/W-v mice indicated that mast cells can contribute to the   ‘ {  aR-dependent release of IL-1.171,172 KitW/W-v mice also did not develop wild-type levels of joint swelling in a model of arthritis elicited by injection of antitype II collagen mAbs followed by LPS. By contrast, KitW-sh/Wsh mice developed features of arthritis in this model that were similar to those of the corresponding wild-type mice.29 *#    # $  # %    ^^   to the autoantibody-induced joint disease may be due to the numbers of neutrophils in these animals. KitW/W-v mice have reduced numbers of bone-marrow and blood neutrophils, whereas KitW-sh/W-sh mice have increased numbers of bone-marrow and blood neutrophils (which may diminish the importance of any contribution of mast cells in this strain). Mast cells have been shown to recruit neutrophils in a mouse model of bullous pemphigoid (an autoimmune disease of the skin) elicited by injection of anti-hemidesmosomal IgG.173 ˆ # # #    "  ^{    ! $ in some models of antibody-mediated diseases. In a model of experimental anti-GBN glomerulonephritis, KitW/W-v mice developed more severe glomerular damage, with more  *^   #     %    !   $   ## mortality, than wild-type mice or mast cell-engrafted KitW/W-v mice.121 Although mast cells are not detectable in the kidneys after induction of this model of glomerulonephritis, mast cell tryptase (mouse transmembrane tryptase [mTMT]) was detected in the draining LNs. *# $ $ # %#! ^{  $   "   #  { #        ƒ~  #     #  ^  #     #   {121 Moreover, there is evidence that at least one mast-cell-derived product, MCPT-4, can promote pathology in this model,174 suggesting that mast cells can have complex roles in this and perhaps

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

203

# $  %#   #   #!  ^{    #  #! ^{  $

OTHER NEGATIVE IMMUNOMODULATORY FUNCTIONS OF MAST CELLS Mast cells also contribute to other models of immunosuppression. Depinay et al175 reported that the bites of Anopheles mosquitoes can impair the development of ^  *^      *`  !$   $   mast cells are present in the bitten skin. How mast cells mediate immunosuppressive function in this model remains to be elucidated. Limon-Flores et al176 used KitW-sh/W-sh mice to show that PGE2 production by mast cells and mast cell migration to draining LNs, were critical for observing the mast-cell-mediated inhibition of CHS responses elicited by epicutaneous application of jet fuel to mouse skin.176 IL-10 also has been implicated as contributing to immunosuppression in this model.176 However, while the jet fuel-induced immunosuppression of CHS in KitW-sh/W-sh   ƒ^•^  <   %   # #   ^^  KitW-sh/W-sh mice, it was not as substantial as that in wild-type mice or in KitW-sh/W-sh mice engrafted with wild-type mast cells. In addition to determining whether mast cells are important sources of IL-10 or PGE2 (or other factors that can mediate immunosuppressive effects) in other examples of immunosuppression, it will be of interest to assess whether mast cells and mast-cell-derived products have important effects on TReg cell numbers, phenotype and/or function in models of CHS or chronic UVB irradiation, or in any of the many other settings in which TReg cells are thought to have an important role.177,178 Studies that investigate the potential interactions between mast cells and TReg are of great interest in helping to unravel the mechanisms of mast-cell-dependent immunosuppression.179,180 *#   !! $  #  % #  ƒ$  109 who showed that mast cells were essential for the optimal induction of peripheral tolerance to skin allografts, which requires the participation of CD4 CD25 FoxP3 TReg cells. TReg cells are a source of IL-9 and IL-9 can mediate the suppression of alloreactive CD8 T cells and act as a mast-cell survival and/or growth factor that can enhance mast-cell function (reviewed in ref. 109). Local production of IL-9 (by TReg cells and/or other sources) may #!  $  # !   # {$ # $    populations within the tolerant allografts. Consistent with the observation that mast cells can promote peripheral tolerance to skin allografts in mice, evidence derived from studies of c-kitt mutant KitWs/Ws versus wild-type rats (in which grafts from the non-inbred KitWs/Ws and wild-type rats were transplanted into KitWs/Ws and wild-type rats, respectively) suggests that mast cells may favor the survival of heterotopic cardiac grafts in rats.181 However, work in C57BL/6-KitW-sh/W-sh versus wild-type mice indicates that the presence or absence of mast cells in the recipient mice (which received cardiac allografts derived from other, mast-cell-containing, mouse strains) makes little or no difference in the features of either an acute or chronic model of cardiac allograft rejection.182 Moreover, local or systemic mast cell degranulation can impair TReg function and lead to the T-cell-dependent acute rejection of established tolerant skin allografts.183 *#       %# # $    !  coculture study showing that mast cells can inhibit TReg suppression, but promote T effector cell expansion and TH17 cell differentiation via an IL-6 and cell proximity dependent

204

MAST CELL BIOLOGY

(OX40/X40L) manner.184 Taken together, the results of both in vivo and in vitro data $ # $       {$ %##   #! ! negative, or neutral effects on allograft survival. In mice, mast cell IgE-dependent effector function can be modulated by TReg. In a mouse model of IgE-mediated passive systemic anaphylaxis, assessment of histamine levels in the serum showed that mast cell activation in response to challenge with IgE     %     !  !$  %^  either in wild-type mice that had been depleted of TReg  !!   ˆ’•^  mice.185 In vitro studies showed that TReg can directly inhibit Fc¡RI-dependent mast cell degranulation (but not mast cell production of IL-6 or TNF) through cell–cell contact involving interactions between OX40 expressed on TReg and OX40 ligand expressed by mast cells.185 *# $    ! *Reg -dependent mechanism, which can suppress mast cell degranulation and which could serve to limit anaphylaxis and perhaps other IgE-dependent responses.

CONCLUSION In summary, mast cells can exert positive immunoregulatory functions in vivo #  # # #      # {   #  cell’s stored mediators and/or cytokines and that can be mediated by functions of mast cells that reside either at the site of the immune response or at peripheral sites, such as within LNs. As outlined above, mast cells can also have negative immunomodulatory functions, in addition to their well-established roles as effector cells. Understanding in detail how individual positive or negative immunomodulatory functions can be induced or suppressed in various mast cell populations will continue to be of considerable interest. `%!  $  #        $$ $  particular immune responses in vivo may be quite challenging. For example, it already is clear that mast cells can have either positive or negative immunomodulatory functions in what would appear to be very similar settings, such as in different mouse models of CHS.7,10,87,186,187 One may even speculate that immune responses will be found in which      # } #  #  # #   #  { # $  %# # # $‹$  "       #   # "   ! # $ { and associated tissue pathology. In support of this hypothesis, both in vitro and in vivo data strongly suggest that one of the mechanisms that promote the mast-cell-dependent IL-10 production that in turn limits certain CHS responses is the activation of mast cells by immune complexes of      24 *# ^     !    mast cell-independent mechanisms, in response to the initial exposure to hapten during the sensitization phase of CHS.24 Thus, in this model, the development of an aspect of the #$    # $   # # ˜ ^  IgG1) results in the generation of a signal (i.e., antigen-IgG1 immune complexes) that    ^{  #  #     #   # local reaction. *#  #   #    # }   phases of an immune response and then help to limit or resolve the local tissue alterations induced by antigen challenge, is consistent with the hypothesis that one key function of

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

205

this cell type is to promote homeostasis—even in those instances when the mast cells also have a major role in perturbing homeostasis in order to promote host defense.17-20  %            #%  $ %##  $ # !  ! $$  $       {$ # ! $  <    ‹$  $  It will also be of interest to assess whether such mast-cell functions can be manipulated to achieve therapeutic ends, such as the enhancement of immune responses that promote health or the suppression of those that result in disease.

REFERENCES 1. Kitamura Y. Heterogeneity of mast cells and phenotypic change between subpopulations. Annu Rev Immunol 1989; 7:59-76. 2. Metcalfe DD, Baram D, Mekori YA. Mast cells. Physiol Rev 1997; 77:1033-79. 3. Kawakami T, Galli SJ. Regulation of mast-cell and basophil function and survival by IgE. Nat Rev Immunol 2002; 2:773-86. 4. Ryan R JJ, Kashyap M, Bailey D et al. Mast cell homeostasis: a fundamental aspect of allergic disease. Crit Rev Immunol 2007; 27:15-32. 5. Marshall JS. Mast-cell responses to pathogens. Nat Rev Immunol 2004; 4:787-99. 6. Galli SJ, Kalesnikoff J, Grimbaldeston MA et al. Mast cells as “tunable” effector and immunoregulatory cells: recent advances. Annu Rev Immunol 2005; 23:749-86. 7. Galli SJ, Nakae S, Tsai M. Mast cells in the development of adaptive immune responses. Nat Immunol 2005; 6:135-42. 8. Abraham SN, St John AL. Mast cell-orchestrated immunity to pathogens. Nat Rev Immunol 2010; 10:440-52. 9. Moon TC, St Laurent CD, Morris KE et al. Advances in mast cell biology: new understanding of heterogeneity and function. Mucosal Immunol 2010; 3:111-28. 10. Grimbaldeston MA, Metz M, Yu M et al. Effector and potential immunoregulatory roles of mast cells in IgE-associated acquired immune responses. Curr Opin Immunol 2006; 18:751-60. 11. Dawicki W, Marshall JS. New and emerging roles for mast cells in host defence. Curr Opin Immunol 2007; 19:31-8. 12. Sayed BA, Brown MA. Mast cells as modulators of T-cell responses. Immunol Rev 2007; 217:53-64. 13. Galli SJ, Grimbaldeston M, Tsai M. Immunomodulatory mast cells: negative, as well as positive, regulators of immunity. Nat Rev Immunol 2008; 8:478-86. 14. Mekori YA, Metcalfe DD. Mast cells in innate immunity. Immunol Rev 2000; 173:131-40. 15. Malaviya R, Abraham SN. Mast cell modulation of immune responses to bacteria. Immunol Rev 2001; 179:16-24. 16. Sayed BA, Christy A, Quirion MR R et al. The master switch: the role of mast cells in autoimmunity and tolerance. Annu Rev Immunol 2008; 26:705-39. 17. Maurer M et al. Mast cells promote homeostasis by limiting endothelin-1-induced toxicity. Nature 2004; 432:512-6. 18. Metz M, Piliponsky AM, Chen CC et al. Mast cells can enhance resistance to snake and honeybee venoms. Science 2006; 313:526-30. 19. Piliponsky AM, Chen CC, Nishimura T et al. Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis. Nat Med 2008; 14:392-8. 20. Schneider LA, Schlenner SM, Feyerabend TB et al. Molecular mechanism of mast cell mediated innate defense against endothelin and snake venom sarafotoxin. J Exp Med 2007; 204:2629-39. 21. Brown JM, Wilson TM, Metcalfe DD. The mast cell and allergic diseases: role in pathogenesis and implications for therapy. Clin Exp Allergy 2008; 38:4-18. 22. Frandji P, Oskéritzian C, Cacaraci F et al. Antigen-dependent stimulation by bone marrow-derived mast cells of MHC class II-restricted T-cell hybridoma. J Immunol 1993; 151:6318-28. 23. Hart PH, Grimbaldeston MA, Swift GJ et al. Dermal mast cells determine susceptibility to ultraviolet B-induced systemic suppression of contact hypersensitivity responses in mice. J Exp Med 1998; 187:2045-53. 24. Grimbaldeston MA, Nakae S, Kalesnikoff J et al. Mast cell-derived interleukin 10 limits skin pathology in contact dermatitis and chronic irradiation with ultraviolet B. Nat Immunol 2007; 8:1095-104. 25. Mills KH. Regulatory T-cells: friend or foe in immunity to infection? Nat Rev Immunol 2004; 4:841-55. _“   @# @@ \<     ^  &^# ^< $ W-sh/W-sh mice as a model for investigating mast cell biology in vivo. Am J Pathol 2005; 167:835-48.

206

MAST CELL BIOLOGY

27. Wolters PJ, Mallen-St Clair J, Lewis CC et al. Tissue-selective mast cell reconstitution and differential lung  "    ^  W-sh/W-sh sash mice. Clin Exp Allergy 2005; 35:82-8. _— ~ ! \  ` > *    !    ^  &sh mice interrupts corin and manifests as hematopoietic and cardiac aberrancy. Am J Pathol 2008; 173:1693-701. _€  #$ >  &           W-sh mice does not impair antibody-mediated arthritis. J Exp Med 2007; 204:2797-802. 30. Piliponsky AM, Chen CC, Grimbaldeston MA et al. Mast cell-derived TNF can exacerbate mortality during severe bacterial infections in C57BL/6-KitW-sh/W-sh mice. Am J Pathol 2010; 176:926-38. 31. Feyerabend TB, Hausser H, Tietz A et al. Loss of histochemical identity in mast cells lacking carboxypeptidase A. Mol Cell Biol 2005; 25:6199-210. 32. K Knight PA, Wright SH, Lawrence CE et al. Delayed expulsion of the nematode Trichinella spiralis in  < # $  ^   $ # $    ^ > ="  2000; 192:1849-56. 33. Pejler G, Abrink M, Ringvall M et al. Mast cell proteases. Adv Immunol 2007; 95:167-255. 34. Thakurdas SM, Melicoff E, Sansores-Garcia L et al. The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections. J Biol Chem 2007; 282:20809-15. 35. Musch W, Wege AK, Mannel DN et al. Generation and characterization of alpha-chymase-Cre transgenic mice. Genesis 2008; 46:163-6. Œ“ # > `    $     ^  @ "\^   !! Transgenic Res 2008; 17:307-15. 37. Feyerabend TB, Terszowski G, Tietz A et al. Deletion of Notch1 converts pro-T cells to dendritic cells and promotes thymic B-cells by cell-extrinsic and cell-intrinsic mechanisms. Immunity 2009; 30:67-79. 38. Newlands GF, Miller HR, MacKellar A et al. Stem cell factor contributes to intestinal mucosal mast cell hyperplasia in rats infected with Nippostrongylus brasiliensis or Trichinella spiralis, but anti-stem cell factor treatment decreases parasite egg production during N. brasiliensis infection. Blood 1995; 86:1968-76. 39. Brandt EB, Strait RT, Hershko D et al. Mast cells are required for experimental oral allergen-induced diarrhea. J Clin Invest 2003; 112:1666-77. 40. Gekara NO, Weiss S. Mast cells initiate early anti-Listeria host defences. Cell Microbiol 2008; 10:225-36. ’  # ~ ‚#    ‚       {  $        upon activation of TLRs by bacteria. Proc Natl Acad Sci USA 2010; 107:8748-53. 42. Soucek L, Lawlor ER, Soto D et al. Mast cells are required for angiogenesis and macroscopic expansion of Myc-induced pancreatic islet tumors. Nat Med 2007; 13:1211-8. 43. Sun J, Sukhova GK, Yang M et al. Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice. J Clin Invest 2007; 117:3359-68. 44. Arumugam T, Ramachandran V, Logsdon CD. Effect of cromolyn on S100P interactions with RAGE and pancreatic cancer growth and invasion in mouse models. J Natl Cancer Inst 2006; 98:1806-18. 45. Norris AA. Pharmacology of sodium cromoglycate. Clin Exp Allergy 26 Suppl 1996; 4:5-7. 46. Blank U, Rivera J. The ins and outs of IgE-dependent mast-cell exocytosis. Trends Immunol 2004; 25:266-73. ’”     *<}< @    #%  ^ ! ~ ‚! $ _••“– 6:218-30. ’—   \! ‚     #      #" >   @ Immunol 2009; 124:639-46; quiz 647-8. ’€ ‚!  >    $ $ !>  @ $ _••“– ”_’^_ 50. Kalesnikoff J, Galli SJ. New developments in mast cell biology. Nat Immunol 2008; 9:1215-23. 51. Gonzalez-Espinosa C, Odom S, Olivera A et al. Preferential signaling and induction of allergy-promoting #< $ %< $  # ##   =     > ="  _••Œ– 197:1453-65. 52. Chong HJ, Andrew Bouton L, Bailey DP et al. IL-4 selectively enhances FcgammaRIII expression and signaling on mouse mast cells. Cell Immunol 2003; 224:65-73. Œ  $ \ >#    ~   ‚ ! $ {^ ‹$$ carcinogenesis. Cancer Cell 2010; 17:121-34. ’ ˆ< š  #$    ="    $ ##^      gamma RI, on human mast cells: up-regulation by IFN-gamma. J Immunol 2000; 164:4332-9.  }    ~<       #      #  { Immunol Rev 2007; 217:304-28. “ ` ž †<  &  *        $    {    ƒ # cells and CTL responses following topical application of TLR7 ligand in mice. Blood 2007; 110:946-53. 57. Heib V, Becker M, Taube C et al. Advances in the understanding of mast cell function. Br J Haematol 2008; 142:683-94. 58. McLachlan JB, Shelburne CP, Hart JP et al. Mast cell activators: a new class of highly effective vaccine adjuvants. Nat Med 2008; 14:536-41.

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

207

59. Hofmann AM, Abraham SN. New roles for mast cells in modulating allergic reactions and immunity against pathogens. Curr Opin Immunol 2009; 21:679-86. 60. Kunder K CA, St John AL, Li G et al. Mast cell-derived particles deliver peripheral signals to remote lymph nodes. J Exp Med 2009; 206:2455-67. 61. McKee AS, Munks MW, MacLeod MK K et al. Alum induces innate immune responses through macrophage      $ #      ‹$   $     ‘$!    immunity. J Immunol 2009; 183:4403-14. 62. Shelburne CP, Nakano H, St John AL et al. Mast cells augment adaptive immunity by orchestrating dendritic    < # $#  $ @ `   _••€– “ŒŒ^’_ 63. K Kulka M, Alexopoulou L, Flavell RA et al. Activation of mast cells by double-stranded RN R A: evidence for activation through Toll-like receptor 3. J Allergy Clin Immunol 2004; 114:174-82. 64. Marshall JS, Jawdat DM. Mast cells in innate immunity. J Allergy Clin Immunol 2004; 114:21-7. “  # > † %  \%< ‚       ž _ @   \## (ed. Pawankar R, Holgate ST, Rosenwasser LJ) 113-133 (Springer Japan, 2009). 66. Supajatura V, Ushio H, Nakao A et al. Differential responses of mast cell Toll-like receptors 2 and 4 in allergy and innate immunity. J Clin Invest 2002; 109:1351-9. 67. Orinska Z, Bulanova E, Budagian V et al. TLR3-induced activation of mast cells modulates CD8 T-cell recruitment. Blood 2005; 106:978-87. 68. Wu L, Feng BS, He SH et al. Bacterial peptidoglycan breaks down intestinal tolerance via mast cell activation: the role of TLR2 and NOD2. Immunol Cell Biol 2007; 85:538-45. 69. Feng BS, He SH, Zheng PY et al. Mast cells play a crucial role in Staphylococcus aureus peptidoglycan-induced diarrhea. Am J Pathol 2007; 171:537-47. 70. Okumura S, Yuki K, Kobayashi R et al. Hyperexpression of NOD2 in intestinal mast cells of Crohn’s      "   {  ^  $ $ ! ~ˆ_   cells. Clin Immunol 2009; 130:175-85. 71. Nakamura Y, Kambe N, Saito M et al. Mast cells mediate neutrophil recruitment and vascular leakage # $# # ~ƒ‚\Œ {  #^ $   > ="  _••€– _•“•Œ”^’“ ”_      *# > *# { $   #  $ ‚! $ _••€– 27:229-65. ”Œ $}  < * ƒ} = {     > @ ! _••€– €Œ•_^ 74. Iwasaki A, Medzhitov R. Regulation of adaptive immunity by the innate immune system. Science 2010; 327:291-5. 75. Gutzmer R, Diestel C, Mommert S et al. Histamine H4 receptor stimulation suppresses IL-12p70 production and mediates chemotaxis in human monocyte-derived dendritic cells. J Immunol 2005; 174:5224-32. 76. Mazzoni A, Young HA, Spitzer JH et al. Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T-cell polarization. J Clin Invest 2001; 108:1865-73. 77. Caron G, Delneste Y, Roelandts E et al. Histamine polarizes human dendritic cells into Th2 cell-promoting effector dendritic cells. J Immunol 2001; 167:3682-6. 78. Mazzoni A, Leifer CA, Mullen GE et al. Cutting edge: histamine inhibits IFN-alpha release from plasmacytoid dendritic cells. J Immunol 2003; 170:2269-73. ”€ %< * %< ~ $ ~   =^!     %#  ^{  factors induce Th2-promoting dendritic cells. Int Immunol 2006; 18:1789-99. 80. Theiner G, Gessner A, Lutz MB. The mast cell mediator PGD2 suppresses IL-12 release by dendritic cells leading to Th2 polarized immune responses in vivo. Immunobiology 2006; 211:463-72. — < \ `< @  ‘     ƒ^_^        #    prostaglandin E 2,promote type 2 cytokine production in maturing human naive T helper cells. J. Immunol, 1997; 159, 28-35. 82. Mazzoni A, Siraganian RP, Leifer CA et al. Dendritic cell modulation by mast cells controls the Th1/Th2 balance in responding T-cells. J Immunol 2006; 177:3577-81. 83. Gosset P, Bureau F, Angeli V et al. Prostaglandin D2 affects the maturation of human monocyte-derived dendritic cells: consequence on the polarization of naive Th cells. J Immunol 2003; 170:4943-52. 84. Gosset P, Pichavant M, Faveeuw C et al. Prostaglandin D2 affects the differentiation and functions of human dendritic cells: impact on the T-cell response. Eur J Immunol 2005; 35:1491-500. 85. Del Prete A, Shao WH, Mitola S et al. Regulation of dendritic cell migration and adaptive immune response by leukotriene B4 receptors: a role for LTB4 in up-regulation of CCR7 expression and function. Blood 2007; 109:626-31. 86. Skokos D, Botros HG, Demeure C et al. Mast cell-derived exosomes induce phenotypic and functional $          $   !! > $ _••Œ– ”•Œ•Œ”^’ 87. Bryce PJ et al. Immune sensitization in the skin is enhanced by antigen-independent effects of IgE. Immunity 2004; 20:381-92.

208

MAST CELL BIOLOGY

88. Jawdat DM, Albert EJ, Rowden G et al. IgE-mediated mast cell activation induces Langerhans cell migration in vivo. J Immunol 2004; 173:5275-82. 89. Suto H, Nakae S, Kakurai M et al. Mast cell-associated TNF promotes dendritic cell migration. J Immunol 2006; 176:4102-12. 90. Jawdat DM, Rowden G, Marshall JS. Mast cells have a pivotal role in TNF-independent lymph node hypertrophy and the mobilization of Langerhans cells in response to bacterial peptidoglycan. J Immunol 2006; 177:1755-62. € %< & >% & $ ~     #  ƒ^“ $ # ! {$"     $   { #  > $ _••– —’_“^_Œ 92. Mekori YA, Metcalfe DD. Mast cell-T-cell interactions. J Allergy Clin Immunol 1999; 104:517-23. 93. Nakae S, Suto H, Kakurai M et al. Mast cells enhance T-cell activation: importance of mast cell-derived TNF. Proc Natl Acad Sci USA 2005; 102:6467-72. 94. K Krishnaswamy G, Lakshman T, Miller AR R et al. Multifunctional cytokine expression by human mast cells: regulation by T-cell membrane contact and glucocorticoids. J Interferon Cytokine Res 1997; 17:167-76. 95. Bhattacharyya SP, Drucker I, Reshef T et al. Activated T-lymphocytes induce degranulation and cytokine production by human mast cells following cell-to-cell contact. J Leukoc Biol 1998; 63:337-41. 96. Inamura N, Mekori YA, Bhattacharyya SP et al. Induction and enhancement of Fc(epsilon)RI-dependent mast cell degranulation following coculture with activated T-cells: dependency on ICAM-1- and leukocyte function-associated antigen (LFA)-1-mediated heterotypic aggregation. J Immunol 1998; 160:4026-33. 97. Baram D, Vaday GG, Salamon P et al. Human mast cells release metalloproteinase-9 on contact with activated T-cells: juxtacrine regulation by TNF-alpha. J Immunol 2001; 167:4008-16. 98. Stopfer P, Mannel DN, Hehlgans T. Lymphotoxin-beta receptor activation by activated T-cells induces cytokine release from mouse bone marrow-derived mast cells. J Immunol 2004; 172:7459-65. 99. Henz BM, Maurer M, Lippert U et al. Mast cells as initiators of immunity and host defense. Exp Dermatol 2001; 10:1-10. •• " @@ >%  &#  @@ ‚          \\^  *^  Cell Immunol 1994; 158:253-64. 101. Poncet P, Arock M, David B. MHC class II-dependent activation of CD4 T-cell hybridomas by human mast cells through superantigen presentation. J Leukoc Biol 1999; 66:105-12. 102. Malaviya R, Twesten NJ, Ross EA et al. Mast cells process bacterial Ags through a phagocytic route for class I MHC presentation to T-cells. J Immunol 1996; 156:1490-6. 103. Stelekati E, Bahri R, D’Orlando O et al. Mast cell-mediated antigen presentation regulates CD8 T-cell effector functions. Immunity 2009; 31:665-76. 104. Gaudenzio N, Espagnolle N, Mars LT et al. Cell-cell cooperation at the T helper cell/mast cell immunological synapse. Blood 2009; 114:4979-88. 105. Kambayashi T, Allenspach EJ, Chang JT et al. Inducible MHC class II expression by mast cells supports effector and regulatory T-cell activation. J Immunol 2009; 182:4686-95. 106. Frandji P, Tkaczyk C, Oskéritzian C et al. Presentation of soluble antigens by mast cells: upregulation by interleukin-4 and granulocyte/macrophage colony-stimulating factor and downregulation by interferon-gamma. Cell Immunol 1995; 163:37-46. 107. Frandji P, Tkaczyk C, Oskeritzian C et al. Exogenous and endogenous antigens are differentially presented by mast cells to CD4 T-lymphocytes. Eur J Immunol 1996; 26:2517-28. 108. Nakano N, Nishiyama C, Yagita H et al. Notch signaling confers antigen-presenting cell functions on mast cells. J Allergy Clin Immunol 2009; 123:74-81 e1. 109. Lu LF, Lind EF, Gondek DC et al. Mast cells are essential intermediaries in regulatory T-cell tolerance. Nature 2006; 442:997-1002. 110. Skokos D, Le Panse S, Villa I et al. Mast cell-dependent B and T-lymphocyte activation is mediated by the secretion of immunologically active exosomes. J. Immunol. 2001; 166:868-76. 111. Kambayashi T, Baranski JD, Baker RG et al. Indirect involvement of allergen-captured mast cells in antigen presentation. Blood 2008; 111:1489-96. _ *<}< @ ž$  †$ š         $  =    $   %^ !   #       $ €€—– €’Œ—^_’ 113. Tkaczyk C, Villa I, Peronet R et al. FcepsilonRI-mediated antigen endocytosis turns interferon-gamma-treated $         ^   $ €€€– €”ŒŒŒ^’• 114. Kashiwakura J, Yokoi H, Saito H et al. T-cell proliferation by direct cross-talk between OX40 ligand on #$    ˆ’•  #$ *^     "     % #$ tonsillar and lung-cultured mast cells. J. Immunol. 2004; 173:5247-57. 115. Nakae S, Suto H, Iikura M et al. Mast cells enhance T-cell activation: importance of mast cell costimulatory molecules and secreted TNF. J Immunol 2006; 176:2238-48.

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

209

116. Xue L, Gyles SL, Wettey FR R et al. Prostaglandin D2 $    $   {  Th2 cytokine production through an action on chemoattractant receptor-like molecule expressed on Th2 cells. J Immunol 2005; 175:6531-6. 117. Jutel M, Watanabe T, Klunker S et al. Histamine regulates T-cell and antibody responses by differential expression of H1 and H2 receptors. Nature 2001; 413:420-5. 118. Wang HW, Tedla N, Lloyd AR R et al. Mast cell activation and migration to lymph nodes during induction of an immune response in mice. J. Clin. Invest. 1998; 102:1617-26. 119. Byrne SN, Limon-Flores AY, Ullrich SE. Mast cell migration from the skin to the draining lymph nodes upon ultraviolet irradiation represents a key step in the induction of immune suppression. J Immunol 2008; 180:4648-55. 120. Tanzola MB, Robbie-Ryan R M, Gutekunst CA et al. Mast cells exert effects outside the central nervous   {$ "    #  $  > $ _••Œ– ”’Œ—^€ 121. Hochegger K, Siebenhaar F, Vielhauer V et al. Role of mast cells in experimental anti-glomerular basement membrane glomerulonephritis. Eur J Immunol 2005; 35:3074-82. 122. Gauchat JF, Henchoz S, Mazzei G et al. Induction of human IgE synthesis in B-cells by mast cells and basophils. Nature 1993; 365:340-3. 123. Tkaczyk C, Frandji P, Botros HG et al. Mouse bone marrow-derived mast cells and mast cell lines constitutively produce B-cell growth and differentiation activities. J Immunol 1996; 157:1720-8. 124. Merluzzi S, Frossi B, Gri G et al. Mast cells enhance proliferation of B lymphocytes and drive their differentiation toward IgA-secreting plasma cells. Blood 2010; 115:2810-7. 125. Echtenacher B, Mannel DN, Hultner L. Critical protective role of mast cells in a model of acute septic peritonitis. Nature 1996; 381:75-7. _“ ! ‚ < * ‚ =     $  $ # {$"       sites of infection through TNF-alpha. Nature 1996; 381:77-80. 127. Nakano, N. et al. Involvement of mast cells in IL-12/23 p40 production is essential for survival from polymicrobial infections. Blood 2007; 109:4846-55. 128. Sutherland RE, Olsen JS, McKinstry A et al. Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by enhancing neutrophil killing. J Immunol 2008; 181:5598-605. 129. Maurer M et al. The c-kit ligand, stem cell factor, can enhance innate immunity through effects on mast cells. J Exp Med 1998; 188:2343-8. 130. Prodeus AP, Zhou X, Maurer M et al. Impaired mast cell-dependent natural immunity in complement @Œ^   ~$  €€”– Œ€•”_^ 131. Gommerman JL, Oh DY, Zhou X et al. A role for CD21/CD35 and CD19 in responses to acute septic peritonitis: a potential mechanism for mast cell activation. J Immunol 2000; 165:6915-21. Œ_  $ š $#  '# `   Œ       !   #   against acute septic peritonitis. J Immunol 2005; 174:4193-7. 133. Orinska Z, Maurer M, Mirghomizadeh F et al. IL-15 constrains mast cell-dependent antibacterial defenses by suppressing chymase activities. Nat Med 2007; 13:927-34. 134. Metz M, Siebenhaar F, Maurer M. Mast cell functions in the innate skin immune system. Immunobiology 2008; 213:251-60. 135. Sher A, Hein A, Moser G et al. Complement receptors promote the phagocytosis of bacteria by rat peritoneal mast cells. Lab Invest 1979; 41:490-9. 136. Malaviya R, Ross EA, MacGregor JI et al. Mast cell phagocytosis of FimH-expressing enterobacteria. J Immunol 1994; 152:1907-14. 137. Di Nardo A, Vitiello A, Gallo RL. Cutting edge: mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide. J Immunol 2003; 170:2274-8. 138. von Köckritz-Blickwede M, Goldmann O et al. Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation. Blood 2008; 111:3070-80. 139. McLachlan JB, Hart JP, Pizzo SV et al. Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection. Nat Immunol 2003; 4:1199-205. 140. Demeure CE, Brahimi K, Hacini F et al. Anopheles mosquito bites activate cutaneous mast cells leading    {    #  #  > $ _••– ”’Œ€Œ_^’• 141. Mallen-St Clair J, Pham CT, Villalta SA et al. Mast cell dipeptidyl peptidase I mediates survival from sepsis. J Clin Invest 2004; 113:628-34. 142. Metz M, Maurer M. Innate immunity and allergy in the skin. Curr Opin Immunol 2009; 21:687-93. 143. Maurer M, Lopez Kostka S, Siebenhaar F et al. Skin mast cells control T-cell-dependent host defense in Leishmania major infections. FASEB J 2006; 20:2460-7. 144. Shin K, Watts GF, Oettgen HC et al. Mouse mast cell tryptase mMCP-6 is a critical link between adaptive and innate immunity in the chronic phase of Trichinella spiralis infection. J Immunol 2008; 180:4885-91. 145. Galli SJ, Tsai M. Mast cells in allergy and infection: versatile effector and regulatory cells in innate and acquired immunity. Eur J Immunol 2010; 40:1843-1851.

210

MAST CELL BIOLOGY

146. Bischoff SC. Role of mast cells in allergic and non-allergic immune responses: comparison of human and murine data. Nat Rev Immunol 2007; 7:93-104. ’” *<< ` '# ` ~      $  {  <  $ from murine mast cells by monomeric IgE and toll-like receptor ligands. Biochem Biophys Res Commun 2010; 391:471-6. 148. Qiao H, Andrade MV, Lisboa FA et al. FcepsilonR1 and toll-like receptors mediate synergistic signals to  < $  $  {  <  $    † _••“– •”“•^— 149. Hart PH, Townley SL, Grimbaldeston MA et al. Mast cells, neuropeptides, histamine and prostaglandins in UV-induced systemic immunosuppression. Methods 2002; 28:79-89. • }  ƒ ž  †   {  $  <   'ž^† #     dependent on endothelin-1 and mast cells. Am J Pathol 2006; 169:815-22. 151. Biggs L et al. Evidence that vitamin D3 promotes mast cell-dependent reduction of chronic UVB-induced skin pathology in mice. J Exp Med 2010; 207:455-63. 152. Alard P, Niizeki H, Hanninen L et al. Local ultraviolet B irradiation impairs contact hypersensitivity induction by triggering release of tumor necrosis factor-alpha from mast cells. Involvement of mast cells and Langerhans cells in susceptibility to ultraviolet B. J Invest Dermatol 1999; 113:983-90. 153. Secor VH, Secor WE, Gutekunst CA et al. Mast cells are essential for early onset and severe disease in a murine model of multiple sclerosis. J Exp Med 2000; 191:813-22. 154. Robbie-Ryan R M, Tanzola MB, Secor VH et al. Cutting edge: both activating and inhibitory Fc receptors expressed on mast cells regulate experimental allergic encephalomyelitis disease severity. J Immunol 2003; 170:1630-4.     ‚‘$  &      ƒ^’ "   $  <   {$ encephalitogenic Th1 responses in EAE. J Clin Invest 2006; 116:1327-36. 156. Sayed BA, Christy AL, Walker ME et al. Meningeal mast cells affect early T-cell central nervous system     ^      # $# *~    $ #  $› > $ 2010; 184:6891-900. 157. Williams CM, Galli SJ. The diverse potential effector and immunoregulatory roles of mast cells in allergic disease. J Allergy Clin Immunol 2000; 105:847-59. 158. Yu M, Tsai M, Tam SY et al. Mast cells can promote the development of multiple features of chronic asthma in mice. J Clin Invest 2006; 116:1633-41. € & @   >      % !  $   #  {  an asthma model in mice. J Exp Med 2000; 192:455-62. 160. Taube C, Wei X, Swasey CH et al. Mast cells, FcepsilonRI and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen exposure in the absence of adjuvant. J Immunol 2004; 172:6398-406. “ ~<  ` ƒ` š$    ^ ! *~  $   % # ! { and TH2 cytokine production in an asthma model in mice. J Allergy Clin Immunol 2007; 120:48-55. 162. Kim YS, Ko HM, Kang NI et al. Mast cells play a key role in the development of late airway hyperresponsiveness through TNF-alpha in a murine model of asthma. Eur J Immunol 2007; 37:1107-15. 163. Reuter S, Heinz A, Sieren M et al. Mast cell-derived tumour necrosis factor is essential for allergic airway disease. Eur Respir J 2008; 31:773-82. 164. Goodarzi K, Goodarzi M, Tager AM et al. Leukotriene B4 and BLT1 control cytotoxic effector T-cell  $  { $ ~ $ _••Œ– ’€“^”Œ 165. Tager AM, Bromley SK, Medoff BD et al. Leukotriene B4 receptor BLT1 mediates early effector T-cell recruitment. Nat Immunol 2003; 4:982-90. 166. Ott VL, Cambier JC, Kappler J et al. Mast cell-dependent migration of effector CD8 T-cells through production of leukotriene B4. Nat Immunol 2003; 4:974-81. 167. Nakae S, Suto H, Berry GJ et al. Mast cell-derived TNF can promote Th17 cell-dependent neutrophil recruitment in ovalbumin-challenged OTII mice. Blood 2007; 109:3640-8. 168. Waern I, Jonasson S, Hjoberg H J et al. Mouse mast cell protease 4 is the major chymase in murine airways  #   !      % { > $ _••€– —Œ“Œ“€^”“ “€ <$     ‚ $ `    ^ ! $        !  elongation in the skin during contact hypersensitivity in mice. Am J Pathol 2006; 169:1713-21. 170. Norman MU, Hwang J, Hulliger S et al. Mast cells regulate the magnitude and the cytokine microenvironment of the contact hypersensitivity response. Am J Pathol 2008; 172:1638-49. ” ƒ      $ #       $ < % $  {  arthritis. Science 2002; 297:1689-92. 172. Nigrovic PA, Binstadt BA, Monach PA et al. Mast cells contribute to initiation of autoantibody-mediated arthritis via IL-1. Proc Natl Acad Sci USA 2007; 104:2325-30. 173. Chen R, Ning G, Zhao ML et al. Mast cells play a key role in neutrophil recruitment in experimental bullous pemphigoid. J Clin Invest 2001; 108:1151-8.

& $ % +

)%(*$ )&+(%k+

)%( ()&+(%

211

174. Scandiuzzi L, Beghdadi W, Daugas E et al. Mouse mast cell protease-4 deteriorates renal function by  $  {     $ "^  $#  > $ 2010; 185:624-33. ”  ~ `  †# &    ^ %^ $  ^  $ responses by mosquito bites. J Immunol 2006; 176:4141-6. 176. Limon-Flores AY, Chacon-Salinas R, Ramos G et al. Mast cells mediate the immune suppression induced by dermal exposure to JP-8 jet fuel. Toxicol Sci 2009; 112:144-52. 177. Beissert S, Schwarz A, Schwarz T. Regulatory T-cells. J Invest Dermatol 2006; 126:15-24. 178. O’Garra A, Vieira P. TH1 cells control themselves by producing interleukin-10. Nat Rev Immunol 2007; 7:425-8. 179. Frossi B, Gri G, Tripodo C et al. Exploring a regulatory role for mast cells: ‘MCregs’? Trends Immunol 2010; 31:97-102. —• ` #< š ‚!  >    *^ $–       ! $ Immunol Lett 2010; 128:98-104. — †    &\ `   {$     $  #    transplantation in rats. Transpl Int 2007; 20:256-65. 182. Itoh S, Nakae S, Velotta JB et al. The role of recipient mast cells in acute and chronic cardiac allograft rejection in C57BL/6-KitW-sh/W-sh mice. J Heart Lung Transplant 2010; 29:401-9. 183. de Vries VC, Wasiuk A, Bennett KA et al. Mast cell degranulation breaks peripheral tolerance. Am J Transplant 2009; 9:2270-80. 184. Piconese S, Gri G, Tripodo C et al. Mast cells counteract regulatory T-cell suppression through interleukin-6 and OX40/OX40L axis toward Th17-cell differentiation. Blood 2009; 114:2639-48. 185. Gri G, Piconese S, Frossi B et al. CD4 CD25 regulatory T-cells suppress mast cell degranulation and allergic responses through OX40-OX40L interaction. Immunity 2008; 29:771-81. 186. Askenase PW, Van Loveren H, Kraeuter K -Kops S et al. Defective elicitation of delayed-type hypersensitivity in W/Wv and Sl/Sld  ^   > $ €—Œ– Œ_“—”^€’ 187. Biedermann T, K Kneilling M, Mailhammer R et al. Mast cells control neutrophil recruitment during T-cell-mediated delayed-type hypersensitivity reactions through tumor necrosis factor and macrophage {    _ > ="  _•••– €_’’^_

CHAPTER R 12 MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS George H. Caughey Department of Medicine, University of California at San Francisco, San Francisco, Califonia, USA    ^< 

Abstract:

Proteases are the most abundant class of proteins produced by mast cells. Many of these are stored in membrane-enclosed intracellular granules until liberated by  $ $ %## $  ^<  ##   =  c¡RI by IgE bound to multivalent allergen. Understanding and separating the functions of the proteases is important because expression differs among mast cells in different tissue locations. Differences between laboratory animals and humans in protease expression  {$ #      %# %## $        $   "   #$ *# {        % #    #    "   # received the most attention, in part because some of them, notably tryptases and chymases, are biomarkers of local and systemic mast cell degranulation and #" #$#   #    $   { and are potential targets for inhibition to treat asthma and related allergic disorders, #    !  ! ^{      "  cell tryptases may protect from serious bacterial lung infections and may limit the ‰ $ Š   { $  ! $   # < Chymases help to maintain intestinal barrier function and to expel parasitic worms and may support blood pressure during anaphylaxis by generating angiotensin II. In other life-or-death examples, carboxypeptidase A3 and other mast cell peptidases limit systemic toxicity of endogenous peptides like endothelin and neurotensin during septic peritonitis and inactivate venom-associated peptides. On the other hand, mast cell peptidase-mediated destruction of protective cytokines, like IL-6, can enhance      \       {$  cell proteases, such as by activating matrix metalloproteinase cascades, which are important in responses to infection and resolution of tissue injury. Overall, mast cell   #!  !    {   ^{   ! and deleterious, in keeping with the increasingly well-appreciated contributions of mast cells in allergy, tissue homeostasis and innate immunity.

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 212

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

213

INTRODUCTION Several recent reviews provide in-depth coverage of particulars of mast cell protease biochemistry, genetics and biological function.1-4 The present chapter emphasizes mast cell protease function as it relates to host defense and its frequent by-product, { *# < %      {   ˜  # $™ $– #  # !     ^{   # *# #   ‘$    # % <    #  %## # "   #  !   $  % $ } ‘ 

 # $       $    $  pathobiology.

PROTECTIVE AND ANTI-INFLAMMATORY EFFECTS   % +`##J &;  @*    & *  ! A classical response to injury (such as a that created by stroking skin with a blunt  $™        #   ‘$  %    {   redness surrounding the region of injury and a wheal due to edema at and near the site injury. This is the so-called triple response of Lewis. The redness or “rubor” aspect is      {  %# ‰ Š ˜#™  $ ˜%™ *#      $  #   #     $ # {$ of the neuropeptide substance P discharged from sensory nerves stimulated by the ‘$  *# {    $      ^   (CGRP) from sensory nerves stimulated by antidromic propagation of sensory nerve signals originating at the site of injury. The transience of CGRP-induced vasodilation is thought to be due to extracellular release of peptidases (Table 1) from mast cells activated by substance P.5 CGRP is hydrolyzed by tryptases and, kinetically speaking,   #  $  $      #$ `-tryptase,6 which inactivates CGRP’s vasodilating actions.7 More recent evidence suggests that primary spinal afferent neurons containing CGRP and substance P also contain protease-activated receptor (PAR)-2,8 which can be activated by tryptases.9-14 Although tryptase is weak compared to trypsin as a PAR-2 activator, mast cells are often near neurons,15 which may be exposed to high concentrations of tryptase during mast cell degranulation. Thus nerves, substance P, CGRP and tryptases may be involved in feedback loops that  $  {     " @ ‚\ %##   # # ^  "       $    $ % in connection with venoms and toxic nonneural peptides. &  " %  J Roles of Tryptases and PAR-2 Tryptase-activated neural PAR-2 is implicated in the component of itching in atopic dermatitis that is unresponsive to antihistamines.16,17 Furthermore, a recently appreciated phenotype of PAR-2-null mice is lowered nociceptive thresholds, such as

214

MAST CELL BIOLOGY

Table 1. Comparison of some mast cell proteases in mice and humans Protease Class

Human

Mouse

SERINE T Tryptase -like Active:

`I, `II, `III, a

 @\^“ ^”| a;  @\^||  @\^”|

Inactive: Chymase-like Active: Inactive: Plasminogen activator CYSTEINE/THIOL Cathepsins METALLO Carboxypeptidase Matrix metallo ADAM, other ASPART P YL

_, b, `III FS– 

CMA-1/_ Cathepsin G t-PA P

 @\^^’^|||– Cathepsin G mMCP-2 ?

DPPI/C, ?others

DPPI/C, B, L, S

CPA P 3 MMP-9 ? Renin

CPA P 3 MMP-9 ADAM17, neurolysin Renin

|  "           || "      # |||   # 

those involving sensitivity to dermal and visceral pain,18 although a role for tryptases in setting pain thresholds in vivo is partly speculative at this point. Itch and pain are both essentially protective, because they alert the host to the presence of pathogens or impending tissue damage. PAR-2 also is expressed in nonneural tissues and cells, such as airway smooth muscle;11 indeed, isolated bronchi constrict in response to PAR-2 agonists, including tryptases.19-22 Nonetheless, it is not yet clear that the bronchoconstricting activities of tryptases are mediated fully via interactions with \‚^_    $  #   # #   # muscle on its own, potentiates the actions of known constrictors, such as histamine, so that they act at lower concentrations and to greater maximum effect.19,20,22,23 Furthermore, PAR-2 agonists acting on epithelium rather than smooth muscle can cause bronchodilation by stimulating epithelial release of PGE2.24 Alternative mechanisms by which tryptase may promote bronchoconstriction include degrading bronchodilating peptides (leaving bronchoconstrictors unopposed),6,25-27 spreading degranulation signals,23 untethering muscle from the bronchial wall by cleaving extracellular matrix or activating matrix-cleaving proteases28 and desensitizing smooth muscle cells to bronchorelaxing `-adrenergic agonists.29 Tryptase’s overall effect is to promote bronchial hyperresponsiveness, a hallmark of asthma. In essence, this is protective, if one accepts that a major purpose of airway smooth muscle is to guard airways from    $% {  $  # "$ $  %##      $   # $ $   $     % # muscle are unknown. Clearly, the purpose is not to cause asthma.

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

215

Figure 1. Mast cell-mediated protection from toxic endogenous peptides in sepsis. Bacterial sepsis is associated with release of toxic peptides like neurotensin and endothelin-1 from nerves and vascular endothelium, respectively. These peptides are recognized by receptors on mast cells, stimulating release, activation or upregulation of detoxifying peptidases, such as carboxypeptidase A3 (CPA3), chymases and neurolysin. This is an example of a homeostatic function of mast cells.

^8> ]   % 8; %;  8]; Neurotensin is a 13-residue fragment of a much larger polypeptide that includes the neuromedin N. Neurotensin gains partial protection from degradation by posttranslational cyclization of its N-terminal amino acid to pyro-glutamate, thereby eliminating its free amino terminus and reducing its sensitivity to shortening by amino-peptidases. ~$   $$     $         $  It causes hypotension when injected into mice; moreover, endogenous neurotensin can {$     #< $  < $     <  survive septic peritonitis. Serum levels of neurotensin are elevated in humans with shock and sepsis. The origin of serum and peritoneal neurotensin is suspected to be neural; however, the actual sources in these conditions are not known.30 Intriguing experiments in mice subjected to cecal ligation and puncture, which models septic peritonitis from a ruptured appendix, suggest that mast cells respond to neurotensin in the peritoneum    "    $#   $ !! #$# $  is not a strong mast cell degranulator, the detoxifying effect of mast cells depends at

216

MAST CELL BIOLOGY

least partially on mast cell expression of neurotensin receptor 1, which may signal these cells to express an inactivating metallopeptidase, neurolysin—possibly on the cell surface (see Fig. 1). Secreted carboxypeptidase A3 also appears to play a role, presumably by removing residues from neurotensin’s unprotected C-terminus. These

 $  "  # <%         mast cells in recovering from cecal ligation and puncture. Whether mast cell-mediated "   $      #     #<   humans, remains to be determined. ]   $ %    Endothelin Many endogenous peptides have the potential to harm as well as to help when produced in response to microbial invasion or tissue injury. One particularly closely examined example, intriguing both in regard to its complexity and physiological importance, is provided by endothelins, which are family of peptides produced by endothelial cells in response to injurious stimuli. Mature endothelins act by engaging receptors on the surface a variety of cells, including vascular smooth muscle and mast cells. They can have acute and longer-term effects, including vasoconstriction, mast cell activation and vascular remodeling. Drugs antagonizing interactions of endothelins with their receptors have found a place, for example, in treating idiopathic pulmonary hypertension. Maurer and colleagues31 described an intriguing nexus between endothelin and mast cells in the peritoneum (see Fig. 1). Endothelin injected into the peritoneal cavity of mice is toxic   $   #   # "  %#  $ $  the context of septic peritonitis. Mouse peritoneal mast cells possess Type A endothelin receptors and respond to endothelin by releasing destructive peptidases, which may include chymases and mast cell carboxypeptidase A3. Schneider and colleagues established more recently that removal of a single tryptophan residue from endothelin’s C-terminus is the principal inactivating event32 in mice. Although other mast cell peptidases, including mouse chymases, can nick endothelin internally, this is not necessarily inactivating, $  } $    # !   $  #  # #  $     ^  $  # can process and activate endothelins from larger, precursor forms. For example, human chymase processes precursor “big” endothelins into a novel, bronchoconstricting form (endothelin [1-31]),33 whereas rat chymases are less selective and degrade big endothelins as well as endothelin [1-31], which possesses bronchoconstricting, vasoconstricting and vascular smooth muscle-proliferating activity33,34 and thus has potential to contribute to pathological bronchoconstriction in human asthma and vascular remodeling in pulmonary hypertension, systemic atherosclerosis and restenosis after angioplasty.         #$   # # !    #^ in asthma-like bronchoconstriction is not clear, since endothelin-1-null mice exhibit airway hyper-responsiveness.35 In humans, then, it appears that mast cells plausibly are involved in activating or inactivating endothelin (or both in succession, since activator chymase and inactivator carboxypeptidase A3 are usually but not inevitably found in the same subsets of mast cells and released together).36-38 Thus, it is reasonable to hypothesize that the role of human mast cells activated by already-mature endothelin is to inactivate the peptide, i.e., to limit toxicity by preventing accumulation and shortening

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

217

duration of action. However, the role of human mast cells activated by other stimuli, $#        ! ! #^     $  big endothelins to smaller active forms, followed perhaps by mast cell carboxypeptidase A3-mediated inactivation. Cathelicidin In studies of human lung mast cells, Schiemann and colleagues showed that the antibacterial peptide, cathelicidin LL-37 (production of which is induced during {™ $     $    `-tryptase, which in turn inactivates cathelicidin.39 This is an example of immunomodulatory negative feedback similar to that described for endothelin. On the other hand, human chymase and cathepsin G in skin mast cells can activate platelet-derived connective tissue activating peptide III to generate neutrophil-activating peptide II, a neutrophil chemokine.40 Therefore, %    #$    $  { %   the nature of mast cell stimulus, the protease phenotype of the stimulated mast cell and the local availability of targets cleavable by the secreted proteases. Cytokines and Interleukins The ability of mast cells to degrade cytokines can be striking. In studies of activated human skin mast cells, exocytosed proteases markedly diminish immunodetection of mast cell-secreted cytokines like IL-6, IL-13 and TNF_, presumably by destroying epitopes recognized by antibodies.41 Inhibitor screens suggest that chymase and cathepsin G are    #     *#    <   ^{   # " # # $  !  %  $ !  {  cytokines. Unless countered by inhibitors, this peptidase activity causes considerable underestimation of mast cell production of several cytokines. The in vivo importance of these effects is less clear, because chymases and cathepsin G are released into environments rich in inhibitors, such as _2-macroglobulin and _1-antichymotrypsin.42-44 There are, however, #    # $  < !       vivo. In septic peritonitis, hydrolytic inactivation of mast cell-derived IL-6 by mMCP-6 tryptase (and perhaps other peptidases) appears to lead to increased mortality.45 This suggests that cytokine-cleaving activities of mast cell proteases can be counterproductive, ! %# $ { ]   >   '# Mast cells have a well-deserved reputation for contributing to severe anaphylactic reactions to bee and wasp stings. Numerous deaths result from systemic release of mast     #<  { #$# #  !   " mixtures of peptides and proteins, some of which directly degranulate mast cells, severe reactions are mediated by mast cell-bound IgE recognizing venom components based on prior sensitization. Because of this association between mast cells and fatal reactions to envenomation, recent evidence46 that mast cells protect mice from lethality of some !     $ ^$! ˆ " %  !   "  class of venoms produced by snakes related to mole vipers. Sarafotoxins are homologous to endothelins, which, as discussed earlier, activate mast cells to release peptidases. Metz

218

MAST CELL BIOLOGY

and colleagues46 #% #   "    " !!  mechanisms and affords some protection from lethal outcomes of envenomation. As is true for endothelin, carboxypeptidase A3 plays a prominent role in disarming the toxin. It is not yet clear whether this effect applies broadly to other venom peptides and proteins. However, mast cells protect mice from hypothermia and death caused by the venom of two snakes (rattlesnake and copperhead) not containing sarafotoxins. Carboxypeptidase A3 involvement in these cases is not as prominent. Thus, for some venoms, other detoxifying peptidases, like chymases and tryptases, may be important. By unclear mechanisms, mast cells also partially protect mice from effects of honeybee venom,46 which contains mast cell-degranulating apitoxins.  "    ]  <

? _  `  

` < j ' *#  

\  $  {    

\^_ (gelatinase A) and MMP-9 (gelatinase B). Although mast cells and mast cell lines can secrete MMP-2 and MMP-9,47-51 these enzymes also are produced by a variety of other {   %##   <   #  $   { $

\^€  "   $  $ #   !  { $  {$    < %# # $  $ #     %## # well-known and often-assessed capacity of these enzymes to hydrolyze denatured collagen (gelatin) in polyacrylamide gels mirrors or predicts their roles in vivo, for these enzymes cleave a variety of proteins52,53 and there is perhaps no equivalent of gelatin in vivo. MMP-9-null mice have phenotypic abnormalities involving long bone angiogenesis,54 but #          % {    # stimulated by mycoplasma infection and these mice do develop neutrophilic pneumonia.55 Although MMP-2 (which increases in lungs of infected MMP-9-null mice) potentially could compensate for lack of MMP-9, MMP-2/MMP-9 double knockout mice have a neutrophilic pneumonia and airway angiogenesis phenotype similar to that of MMP-9-null mice.55     $ #   $  {    # regard53,56,57 because lack of MMP-2 and, especially of MMP-9, hinders egress of recruited eosinophils and other leukocytes from pulmonary interstitium into the airspaces, from which they would otherwise be cleared via the mucociliary and/or macrophage-mediated apoptotic pathways. This clogs interstitial spaces with leukocytes, impairs gas transfer and ‰#"Š #  *#     $ $ # $ #

\^_ 

\^€   $    {  $  #   {  $ #    {    may be an important function of these MMPs, as in a model of bronchopulmonary dysplasia, in which transgenic Mmp9
MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

219

inhibitor tissue inhibitor of metalloproteinases (TIMP)-1. Thus, MMP-9 is secreted as an inactive, pro-MMP-9/TIMP-1 complex.47,60 Activation of the mast cell-secreted complex occurs outside of the cell, especially in cells that are stimulated to release contents of serine protease-rich secretory granules. Because mast cell MMP-9 seems to be secreted independently of its serine protease activators, it is likely that the pro-MMP/TIMP-1 complex originates in a compartment separate from the classic secretory granule. Human mast cells also exhibit the interesting property of secreting MMP-9 upon direct contact with T-lymphocytes.50 Activation, Disinhibition and Destruction of MMPs by Chymases The interactions of mast cell chymases with MMP-9 are biochemically intriguing and possibly unique. Studies of canine mast cell chymase and pro-MMP-9 reveal that chymase activates pro-MMP-9 by selectively hydrolyzing residues in the MMP-9 pro-peptide.47,60 This yields a large gain in solution-phase proteolytic activity. Beyond this, chymase activates MMP-9 even while bound to TIMP-161 and is the only MMP-9 activator shown to possess this capability. Because much extracellular MMP-9 is imbedded in matrix as a TIMP-1-bound pro-enzyme, chymase released from mast cells can bring MMP-9 to life from latency and launch cascades of MMP activation initiated by MMP-9, which can activate other MMPs. Chymase induces the activation of TIMP-1-bound MMP-9 by cleaving TIMP-1 itself. Although interactions between chymase and MMP-2 have been studied less intensively, chymase inhibitors are reported to decrease intimal hyperplasia in balloon-injured canine carotid arteries, with an accompanying decrease in activated MMP-2.62 Although the biochemistry of MMP-9 has been explored in greatest detail using canine enzymes and mast cell lines, the phenomenon occurs in other mammals. In mice, for example, the principal pro-mMMP-9-activating chymase is mMCP-4,49,51 whose actions are hypothesized to be important in tumor growth and invasion in a model of skin cancer49 as well as in $  #<  $   $$ *#    attributed to reduced matrix turnover in mMCP-4-null mice,51 which also suggests that mMCP-4 regulates levels of MMP-2.51 More recently, a study of chymase inhibitors in a colitis model revealed markedly reduced colonic MMP-9 levels in inhibitor-treated mice.63 Similar effects occur in angiotensin II-induced aortic aneurysms.64 Chymase can destroy MMP-9 activity with prolonged incubations, although its preference appears to be for activating cleavages. It should be stressed that the net effect of MMP-9 !  # $  ^{  !

\^ۤ !!  $  {

  @^ "   *@ ;# Updating Paradigms Research over the past two decades has built a solid body of evidence supporting physiologically important mast cell peptidase activation of the renin-angiotensin system, whose end product is the vasoactive octapeptide angiotensin II. Several aspects of involvement of mast cell peptidases in generating angiotensin II are at odds with the partly outmoded concept that the key steps in generating angiotensin II occur within vessels, starting with regulated release of the aspartyl peptidase renin

220

MAST CELL BIOLOGY

into the bloodstream by kidney cells responding to drops in renal perfusion pressure. According to the classical paradigm, renin then cleaves off a portion of the N-terminus of a circulating protein, angiotensinogen, to generate the decapeptide angiotensin I. This inactive peptide is then hydrolyzed by angiotensin converting enzyme (ACE), an ecto-metallopeptidase on the surface of pulmonary vascular endothelial cells, to generate the angiotensin II, which binds to receptors in arteriolar smooth muscle to constrict vessels and acts on the adrenal gland and kidney to promote retention of sodium and water. This process is homeostatic, being designed to preserve blood pressure. Additional essentially protective roles may include vasoconstriction to reduce bleeding and edema during tissue injury. In this regard it is interesting to note the association between use of ACE inhibitors and more severe systemic reactions to insect stings.65 ACE and angiotensin II are unquestionably important in human health, as shown by the success of therapeutic agents directed against ACE or receptors of its product, angiotensin II. As discussed in more detail below, mast cells complicate the classical paradigm by providing the means of generating both angiotensin I and II outside of vessels, using pathways that neither involve ACE nor are blocked by ACE inhibitors. Because mast cells outside of the kidney can be a source of renin, they can also initiate angiotensin production in a variety of tissues in response to events unconnected to regulation of blood pressure and volume. Recent research on angiotensins also suggests that angiotensin II is a trophic factor with chronic affects on growth and remodeling of many tissues and that angiotensin II-inactivating peptidases may be important in regulating angiotensin activity. Furthermore, studies suggest that there may be “good” and “bad” receptors for angiotensin II in the context, for example, of responses to lung injury.66,67 Generation of Angiotensin II by Chymases and Cathepsin G The idea that some mast peptidases generate bioactive angiotensin II is not new. Travis, Wintroub and others showed that human mast cell chymase and cathepsin G process angiotensin I into angiotensin II in vitro more than a quarter of a century ago,68,69    # } %   $   ƒ  !   % <  different laboratories culminated in a realization that a potent, non-ACE, angiotensin II-generating peptidase extractable from human heart tissue is, in fact, indistinguishable from human mast cell chymase.70-73 Although some evidence supports the possibility that this chymase is expressed in endothelial, vascular or cardiac muscle cells, it is clear that mast cells have far greater capacity to store chymase than any other cell type. Thus, it is probable that most chymase in extracts of human heart muscle, as in other tissues, arises from MCTC mast cells, which are easily detected in heart tissues. History was repeating itself, in the sense that a similar convergence occurred twice in the 1970’s in connection with the major mast cell chymotryptic protease of rat connective tissue, mast cell protease I, which was initially thought to be intrinsic to skeletal muscle.74,75 Similarly, an enzyme extracted from rat intestine, thought to function to degrade intracellular pyridoxal phosphate-dependent enzymes, was subsequently found to be made and stored by an intestinal mast cell sub-population, then called “atypical”.76 These events in the history of mast cell peptidases are worth recounting if only because they remind us that the capacity of mast cells to store peptidases is prodigious; so much so that a minor cell in a tissue like skeletal muscle can produce the lion’s share of certain peptidase activities in tissue extracts.

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

221

Although human cathepsin G was shown long ago to be capable of selectively hydrolyzing angiotensin I to generate angiotensin II68 and shown later to be an abundant product of MCTC mast cells,77,78 rather little is known of its importance in generating angiotensin II in vivo and even less known of its role in other animals. A preliminary comparison of the properties of mouse and human enzymes suggests that the former is   !   %       $  #   #$ cathepsin G is a major product of neutrophils (and to a lesser extent, macrophages), in addition to being a product of MCTC mast cells. Thus, it may contribute to the generation of angiotensin II in neutrophilic as well as mast cell-mediated pathologies. In this connection, it may be helpful that potent, dual inhibitors of human chymase and cathepsin G have been generated.79,80 In humans, chymase can generate angiotensin II while bound to macroglobulin, which prolongs chymase’s duration of action and escorts chymase away from site of generation, where it can be detected in blood.44 @#     {$   $  $#   <     in this macroglobulin-bound but active form than to be inactivated irreversibly by  ^ #   %## # #    @#   # # hand, is susceptible to _1-antichymotrypsin. In mice, notwithstanding the apparent redundancy of chymase-like peptidases, it appears that mMCP-4 makes the major contribution, as suggested by studies in mMCP-4-null mice.81 However, mMCP-4 is not as selective as human chymase for the Phe8-His9 bond of angiotensin I and is both an activator and inactivator of angiotensin.82 Nonetheless, as established by the work of Husain and Dell’Italia and others, mMCP-4 and/or similar chymases appear to be responsible for ACE inhibitor-resistant generation of angiotensin II in mice and for most extravascular generation of angiotensin II in heart muscle,83 as previously shown in dogs.84 Although there is potential for production of mMCP-4 and other chymases by nonmast cells, the marked reduction of interstitial       ^  KitW/KitW-v mice suggests that most of the extravascular angiotensin II-generating capacity originates from mast cells.83 Presumably, the existence of this extravascular pathway serves a homeostatic purpose, for example the control of blood pressure. However, many studies in this active area of research focus on potentially deleterious effects such as hypertension (as shown in transgenic mouse expressing rat vascular chymase),85    %  lung injury in ARDS and cardiac arrhythmias.83,86-88 Mast Cell Renin Studies by Mackins and colleagues89,90 establish that mast cells can store renin, which is released by mast cell activators like compound 48/80 and allergen. Renin boosts local generation of angiotensin I, which is then processed to angiotensin II by ACE or chymase-like peptidases. Presumably, mast cell renin could serve homeostatic functions, although existing studies focus on potential pathological contributions. In  $ $ #        %#  ‰! Š   and fewer malignant ventricular arrhythmias after ischemia-reperfusion. Intriguingly, much of this work on cardiovascular implications of mast cell renin release has been conducted in guinea pigs,89,90 which appear to lack angiotensin II-generating chymase.89 Whether mast cell-derived renin is important in myocardial ischemia and arrhythmias in humans needs to be established.

222

MAST CELL BIOLOGY

Figure 2. Selff termination of mast cell activation by allergen. Multivalent protein allergens like birch    }   ^  = $  ##^  =   # $    cells stimulates degranulation and release of epitope-destroying proteases, such as chymases. This is a ! <  # $   {     

   !#   =# Perhaps the earliest in vivo evidence that a mast cell peptidase serves host defense %  !    $ %# !  $    # $ intraepithelial mast cell chymase mMCP-1.91 *# % #     to lack a mast cell serine peptidase. These Mcpt1^$  #!  $ " Trichinella spiralis,91 which is a parasitic roundworm that infects a variety of mammalian hosts and causes trichinellosis; a disease characterized most dramatically in humans  $ {   $    !

@\^^"   cells increase dramatically in mice after worm infestation and expulsion of Trichinella spiralis accompanies or precedes mMCP-1 release into intestinal mucosa and lumen. This  $    { ! # #    $  " %  more quickly, restores gut health sooner. Curiously, mMCP-1’s de-worming function does not extend to all nematodes. For example, although mice infected with Nippostrongylus brasiliensis develop impressive jejunal mastocytosis and dramatic increases in gut content

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

223

of mMCP-1, worm burden is unaffected by lack of mMCP-1 in Mcpt1-null mice.91-94 However, other mast cell proteases may be important. Indeed, recent studies suggest that the related `-chymase, mMCP-4, plays a more general role in regulating gut barrier function, including permeability and epithelial migration. This is indicated by the small intestine phenotype in Mcpt4^$ –    ^   %#   populations re-established by adoptive transfer of BMCMC originating from wild-type and Mcpt4
224

MAST CELL BIOLOGY

mast cell proteases could also help to control gut infections was suggested by studies in mice showing that the mouse tryptase, mMCP6, but not the related protease mMCP7, attracts neutrophils when injected into the peritoneal cavity.108 These studies furthermore established that Mcpt6
PRO-INFLAMMATORY ROLES OF MAST CELL PEPTIDASES Tryptases   j ' < $^ 

3" Tryptases are released with histamine from human skin mast cells in acute and chronic in vivo responses to allergen.114,115 Levels of immunoreactive tryptases also increase in the airway in asthma116 and transcripts encoding tryptases are among the most abundant and upregulated gene products in brush biopsies of asthmatic bronchial epithelium, consistent with intraepithelial mast cell migration or local proliferation.38,117 It is not known if tryptases   $   % {$ ˜    #  ™   ! Overall, evidence from animal models as well as humans is compelling that tryptases,  $    {      <     ! $ also contribute to resulting pathology. For example, small molecules designed to inhibit human tryptases markedly reduce airway eosinophilia and goblet cell hyperplasia in a mouse model of asthma.118       %# 119 which is a highly potent, although not entirely selective inhibitor of tryptases.120 Several lines of ! $ #        !!      " nafamostat reduces scratching in mice induced by skin injection of tryptase, or by the mast cell-degranulator compound 48/80.17 Nafamostat’s effects on scratching are not seen in  ^    !! \‚^_  #      $ 48/80 on scratching are inhibited by PAR-2 antagonists. The simplest explanation of

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

225

#   #       ! \‚^_   ! !!  itch pathways. Note that leeches make a potent and selective tryptase inhibitor,121 which is indirect evidence of a role for tryptase in signaling the presence of the leech, perhaps by activating neural itch and pain pathways or by spreading the degranulation signal. Published studies of the effect of tryptase inhibition in humans are few. However, a topical (aerosolized) tryptase inhibitor reduced late-phase bronchoconstriction in a small study involving mild human atopic asthmatics122 and decreased nasal symptoms and eosinophilia in humans with allergic rhinitis.123 Several pathways by which tryptases may promote asthmatic bronchoconstriction have been proposed. However, it is not yet clear which are the most important. One potential mechanism, proposed early on, is the tryptase-mediated destruction of vasoactive intestinal peptide.6,25-27 A possibly distinct pathway involves augmentation of bronchoconstriction by histamine and other airway smooth muscle agonists, which is a phenomenon manifest in muscle bath preparations of bronchi from dogs and humans.19,20,22 Tryptase released from one mast cell under the {$         $       $ by mast cell-stabilizing actions of some tryptase inhibitors124,125 and by the provocation of histamine release in sheep or guinea pig skin or airway by injected or inhaled human tryptase.23,126-129 The early history of pharmaceutical interest in and development of tryptase inhibitors was thoroughly reviewed by Cairns130 and will not be rereviewed here. Notwithstanding the pharmaceutical interest in blocking more acute affects of tryptases on {  # $   # #     %  $  %#   %  131-134 smooth muscle135 and vessels,136 which may be responsible for bronchodilator-resistant airway obstruction, also provide rationales for therapeutic inhibition. The extent to which tryptase activation of PAR-2 is involved    {   #           

j '^ 0$ {          #   $  $ {   ^    %    # 137,138 One of these models (methylated bovine serum albumin/IL-1`-induced) seems to require two tryptase gene products,   @\^“   ^”  $ "   # {  #137 These   $    $ $ #       $   #    $   ^    #    ^"  mast cells in arthritic joints.139-142 The mechanisms by which mast cell tryptases contribute to various forms of experimental arthritis remain to be established and, of course, tryptases and mast cells are not the sole factors contributing to the phenotype.141,143 The          # #   #$ { < #$  #         *# $            {  %   $ !  %## has been long associated with increases in mast cell numbers and activation in affected tissues.144,145 At least two pharmacological lines of evidence suggest that tryptases released from mast cells contribute to the pathology of ulcerative colitis: (1) a human trial of a `-tryptase-selective inhibitor, given systemically by subcutaneous injection, appeared to reduce gastrointestinal symptoms in subjects with ulcerative colitis146 and (2) treatment %# #   #    # !   #   a rat model of colitis caused by trinitrobenzene sulfonic acid.147

226

MAST CELL BIOLOGY

;#     j '  $^ 

3" The ability of human chymase and cathepsin G to cleave angiotensin I selectively at Phe8 to generate angiotensin II which, in addition to being a homeostatic process  $     $      ^{  % $ }    `%!  # } #!  #      {  especially by promoting tissue swelling. For example, dog chymase, injected into dog skin, increases the size of histamine-induced wheals without inducing wheals by itself.148 Furthermore, inhibition of chymase activity in vivo reduces the size of wheals generated by mast cell-degranulating agents. The mechanisms of these effects are not known, but $ $  <%  $#   " $  "  # {$ " ! $ # {$  #  !  # #  %$  # % †# # and cathepsin G are fairly omnivorous and can separate the dermal-epidermal junction by degrading a variety of matrix proteins,149 as well as by activating MMPs.49 Possibly, these enzymes also destroy extracellular histaminases so that histamine levels are higher and more sustained. Chymase and cathepsin G also can stimulate gland secretion.150,151 Indeed, in human airways, chymase-positive mast cells are a high fraction of mast cells lying within 20 +m of submucosal glands.152 The proteolytic activity of human chymase extends to albumin;153 however, this cleavage would not increase oncotic pressure because # <    ‘  $  <  $$  #  #^<      {    $  sponge granulomas in hamsters154 and by mast cell-dependent angiogenesis in a model of skin carcinogenesis.49 The recently reported inhibition of several animal models of    ^  {  #   #  # 80 further $ # # }         { \ they act synergistically when released from human mast cells, where they are usually found in the same granules, because their substrate preferences only partially overlap. j*`< j =^|  ^ 

} 3   $    !  #^  $ ‘$  $ #    mouse mast cell protease, mMCP-5, is responsible for irreversibly injuring skeletal muscle.155 This work establishes the principle that a mast cell protease can be cytotoxic in the context  # { %##   %#   ! *# # by which muscle is damaged by mMCP-5, which is an elastolytic peptidase with no known functional equivalent, in human mast cells is not clear. However, the potential for chymase-related mast cell peptidases to damage and alter vessels themselves has gathered increased experimental support over the past few years. After initial studies suggested a role for mast cells in promoting arterial enlargement in a neutrophil elastase-induced mouse model of aortic aneurysm,156 subsequent studies showed at least partial dependence on mast cell expression of mMCP-4.103 The mechanism by which this chymase promotes $     #   ##}  $ {  ! of vessel wall-weakening MMPs and cathepsins and direct stimulation of aortic smooth muscle cell apoptosis. On the other hand, there is strong in vivo pharmacological evidence from studies in a variety of models of vascular injury that chymase-like enzymes promote  !$         ‘$ 62,88,157-160

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

227

;  +  ( &  Membrane-bound mast cell secretory granules, harboring biogenic amines, proteases and proteoglycans, are related to lysosomes and thus partly may serve lysosomal functions. Indeed they contain some lysosome-associated proteases. Cathepsin G is not a classic cathepsin because it is a serine, not thiol, peptidase with expression restricted to specialized granules of mast cells and myelomonocytic cells, especially neutrophils. Another granule peptidase, dipeptidyl peptidase I (DPPI; cathepsin C) also is atypical. Although it is expressed in many cells, it is much more abundant in mast cells, myelomonocytes and # }  $  < " *  $  <   ${ dog airway, mast cells are the dominant cell type staining positively for DPPI.161 Like other proteins of secretory granules, DPPI can be secreted162 and it may cleave extracellular targets.161 However, it is not highly destructive because its activities are restricted compared to other thiol cathepsins by an “exclusion domain”, ensuring preference for cleaving N-terminal dipeptides.163 These attributes suggest that DPPI is likely to serve an intragranular function not related to general protein degradation or typical lysosomal !    \\§ ‘      !  $^ $ cell peptidases related to chymases, cathepsin G, lymphocyte granzymes and neutrophil elastase. It accomplishes this by removing the N-terminal pro-dipeptide that is a shared attribute of these enzymes.164-167 Given the effects of genetic inactivation of DPPI on activation of an impressive range of conserved immune serine peptidases, the phenotype might be expected to be more severe than it is. In fact, genetic deletion or inactivation  \\   #        $ \\^   #!  !   $        14,168 including improved short term survival following cecal ligation and puncture.45 Humans with defects in DPPI have chronic periodontal infections.169 Mice lacking DPPI have little if any mast cell chymase activity, although at least one chymase (mMCP-4) is present in mast cell granules as an inactive pro-enzyme.166 The effect on mouse tryptase mMCP-6 is less dramatic, with activity being reduced but present.166 This is perhaps not surprising, given that tryptases possess a much longer pro-peptide than chymases, granzymes and neutrophil elastase-like hydrolases.170,171 Schwartz and colleagues suggest that the pro-peptide is removed from human `-tryptases by tandem cleavages initiated by autocatalysis to generate a remnant pro-dipeptide removed by DPPI.172 The extent to which human chymases and tryptases depend on DPPI for activation in vivo remains to be determined. Mast cells do express   # # $ #  %##  {$ ! of chymase and/or carboxypeptidase independently of DPPI in mouse mast cells.173,174 Thus, although DPPI has received more attention in studies of mast cell biology to date, other thiol cathepsins may be important to mast cell function.

CONCLUSION Mast cell peptidases and proteases, particularly those that are stored in secretory  $    "  $  $  {  on the tissue and context in which they are released. Mounting evidence also suggests that they play critical, supporting roles in protective, innate immune responses by ! " $   %# {     peptides in venoms and supporting blood pressure via generation of angiotensin II. These

228

MAST CELL BIOLOGY

contributions and the pathways leading to them remain active areas of basic and clinical investigation. In summary, proteases—which are the major proteins stored and released by mast cells—are immunoregulatory proteins that contribute in a variety of important %  {  ! #   !  $ #

REFERENCES 1. Stevens RL, Adachi R. Protease-proteoglycan complexes of mouse and human mast cells and importance of their `^ ^#  "  {   $ $ ‚! _••”– _”^“” _ @$# `      #  {  #  $ ‚! _••”– 217:141-154. Œ \‘  ‚  = &        ^ $ $   {   Blood 2010 [Epub ahead of print]. 4. Trivedi NN, Caughey GH. Mast cell peptidases: Chameleons of innate immunity and host defense. Am J Respir Cell Mol Biol 2010; 42:257-267. 5. Brain SD, Williams TJ. Substance P regulates the vasodilator activity of calcitonin gene-related peptide. Nature 1988; 335:73-75. 6. Tam EK, Caughey GH. Degradation of airway neuropeptides by human lung tryptase. Am J Respir Cell Mol Biol 1990; 3:27-32. 7. Walls AF, Brain SD, Desai A et al. Human mast cell tryptase attenuates the vasodilator activity of calcitonin gene-related peptide. Biochem Pharmacol 1992; 43:1243-1248. — #  ž  ~ š$ `      ^!  _ $ { by a neurogenic mechanism. Nat Med 2000; 6:151-158. 9. Corvera CU, Dery O, McConalogue K et al. Mast cell tryptase regulates rat colonic myocytes through PAR-2. J Clin Invest 1997; 100:1383-1393. 10. Molino M, Barnathan ES, Numerof R et al. Interactions of mast cell tryptase with thrombin receptors and PAR-2. J Biol Chem 1997; 272:4043-4049. 11. Schmidlin F, Amadesi S, Vidil R et al. Expression and function of PAR-2 in human bronchial smooth muscle. Am J Respir Crit Care Med 2001; 164:1276-1281. 12. Steinhoff M, Corvera CU, Thoma MS et al. PAR-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase. Exp Dermatol 1999; 8:282-294. 13. Compton SJ, Renaux B, Wijesuriya SJ et al. Glycosylation and the activation of PAR-2 by human mast cell tryptase. Br J Pharmacol 2001; 134:705-718. 14. Cottrell GS, Amadesi S, Pikios S et al. PAR-2, dipeptidyl peptidase I and proteases mediate Clostridium   "       _••”– Œ__’__^_’Œ” 15. Stead RH, Dixon MF, Bramwell NH et al. Mast cells are closely apposed to nerves in the human gastrointestinal mucosa. Gastroenterology 1989; 97:575-585. 16. Steinhoff M, Neisius U, Ikoma A et al. PrAR-2 mediates itch: a novel pathway for pruritus in human skin. J Neurosci 2003; 23:6176-6180. 17. Ui H, Andoh T, Lee JB et al. Potent pruritogenic action of tryptase mediated by PAR-2 receptor and its involvement in anti-pruritic effect of nafamostat mesilate in mice. Eur J Pharmacol 2006; 530:172-178. 18. Cenac N, Andrews CN, Holzhausen M et al. Role for protease activity in visceral pain in irritable bowel syndrome. J Clin Invest 2007; 117:636-647. 19. Sekizawa K, Caughey GH, Lazarus SC et al. Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs. J Clin Invest 1989; 83:175-179. 20. Johnson PRA, Ammit AJ, Carlin SM et al. Mast cell tryptase potentiates histamine-induced contraction in human sensitized bronchus. Eur Resp J 1997; 10:38-43. 21. Barrios VE, Middleton SC, Kashem MA et al. Tryptase mediates hyperresponsiveness in isolated guinea pig bronchi Life Sci 1998; 63:2295-2303. 22. Berger P, Compton SJ, Molimard M et al. Mast cell tryptase as a mediator of hyperresponsiveness in human isolated bronchi. Clin Exp Allergy 1999; 29:804-812. 23. Molinari JF, Scuri M, Moore WR R et al. Inhaled tryptase causes bronchoconstriction in sheep via histamine release. Am J Respir Crit Care Med 1996; 154:649-653. 24. Cocks TM, Fong B, Chow JM et al. A protective role for protease-activated receptors in the airways. Nature 1999; 398:156-160. 25. Caughey GH, Leidig F, Viro NF et al. Substance P and vasoactive intestinal peptide degradation by mast cell tryptase and chymase. J Pharmacol Exp Ther 1988; 244:133-137.

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

229

26. Franconi GM, Graf PD, Lazarus SC et al. Mast cell chymase and tryptase reverse airway smooth muscle relaxation induced by vasoactive intestinal peptide in the ferret. J Pharmacol Exp Ther 1989; 248:947-951. 27. Tam EK, Franconi GM, Nadel JA et al. Protease inhibitors potentiate smooth muscle relaxation induced by vasoactive intestinal peptide in isolated human bronchi. Am J Respir Cell Mol Biol 1990; 2:449-452. 28. Gruber BL, Marchese MJ, Suzuki K et al. Synovial procollagenase activation by human mast cell tryptase. J Clin Invest 1989; 84:1657-1662. 29. Kobayashi M, Kume K H, Oguma T et al. Mast cell tryptase causes homologous desensitization of `-adrenoceptors by Ca2 sensitization in tracheal smooth muscle. Clin Exp Allergy 2008; 38:135-144. 30. Piliponsky AM, Chen CC, Nishimura T et al. Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis. Nat Med 2008; 14:392-398. 31. Maurer M, Wedemeyer J, Metz M et al. Mast cells promote homeostasis by limiting endothelin-1-induced toxicity. Nature 2004; 432:512-516. 32. Schneider LA, Schlenner SM, Feyerabend TB et al. Molecular mechanism of mast cell mediated innate defense against endothelin and snake venom sarafotoxin. J Exp Med 2007; 204:2629-2639. 33. Nakano A, Kishi F, Minami K et al. Selective conversion of big endothelins to tracheal smooth muscle-constricting 31-amino acid-length endothelins by chymase from human mast cells. J Immunol 1997; 159:1987-1992. 34. Nagata N, Niwa Y, Nakaya Y. A novel 31-amino-acid-length endothelin, ET-1(1-31), can act as a biologically active peptide for vascular smooth muscle cells. Biochem Biophys Res Commun 2000; 275:595-600. 35. Nagase T, Kurihara K H, K Kurihara Y et al. Airway hyperresponsiveness to methacholine in mutant mice    #^  > ‚ @  @   €€—– ”“•^“’ 36. Irani AM, Goldstein SM, Wintroub BU et al. Human mast cell carboxypeptidase: Selective localization to MCTC cells. J Immunol 1991; 147:247-253. 37. Goldstein SM, Leong J, Schwartz LB et al. Protease composition of exocytosed human skin mast cell protease-proteoglycan complexes: tryptase resides in a complex distinct from chymase and carboxypeptidase. J Immunol 1992; 148:2475-2482. 38. Dougherty RH, Sidhu SS, Raman K et al. Accumulation of intraepithelial mast cells with a unique protease phenotype in Th2-high asthma. J Allergy Clin Immunol 2010; 125:1046-1053. 39. Schiemann F, Brandt E, Gross R et al. The cathelicidin LL-37 activates human mast cells and is degraded by mast cell tryptase: counter-regulation by CXCL4. J Immunol 2009; 183:2223-2231. 40. Schiemann F, Grimm TA, Hoch J et al. Mast cells and neutrophils proteolytically activate chemokine precursor CTAP-III and are subject to counterregulation by PF-4 through inhibition of chymase and cathepsin G. Blood 2006; 107:2234-2242. 41. Zhao W, Oskeritzian CA, Pozez AL et al. Cytokine production by skin-derived mast cells: endogenous proteases are responsible for degradation of cytokines. J Immunol 2005; 175:2635-2642. 42. Schechter NM, Sprows JL, Schoenberger OL et al. Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors. J Biol Chem 1989; 264:21308-21315. ’Œ &  $ ‚  ## ~  `#   #  #$ #  _2-macroglobulin. Arch Biochem Biophys 1999; 368:276-284. 44. Raymond WW, Su S, Makarova A et al. _2-Macroglobulin capture allows detection of mast cell chymase in serum and creates a reservoir of angiotensin II-generating activity. J Immunol 2009; 182:5770-5777. 45. Mallen-St Clair J, Pham CT, Villalta SA et al. Mast cell dipeptidyl peptidase I mediates survival from sepsis. J Clin Invest 2004; 113:628-634. 46. Metz M, Piliponsky AM, Chen CC et al. Mast cells can enhance resistance to snake and honeybee venoms. Science 2006; 313:526-530. 47. Fang KC, Raymond WW, Lazarus SC et al. Dog mastocytoma cells secrete a 92-kd gelatinase activated extracellularly by mast cell chymase. J Clin Invest 1996; 97:1589-1596. 48. Fang KC, Wolters PJ, Steinhoff M et al. Mast cell expression of gelatinases A and B is regulated by kit ligand and TGF-`. J Immunol 1999; 162:5528-5535. ’€ @$ ƒ  ‚ && †     {    $ $  $  squamous epithelial carcinogenesis. Genes Dev 1999; 13:1382-1397. 50. Baram D, Vaday GG, Salamon P et al. Human mast cells release metalloproteinase-9 on contact with activated T-cells: juxtacrine regulation by TNF_. J Immunol 2001; 167:4008-4016. 51. Tchougounova E, Lundequist A, Fajardo I et al. A key role for mast cell chymase in the activation of proMMP-9 and proMMP-2. J Biol Chem 2005; 280:9291-9296. _ Ÿ$   >` * =   {     !   chemoattractant protein-3. Science 2000; 289:1202-1206. Œ  > @  † =    \      !! $  

\ _  € !  #  $  { > $ _••“– ”””Œ_^”Œ_ 54. Vu TH, Shipley JM, Bergers G et al. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 1998; 93:411-422.

230

MAST CELL BIOLOGY

55. Baluk P, Raymond WW, Ator E et al. Matrix metalloproteinase-2 and -9 expression increases in mycoplasma-infected airways but is not required for microvascular remodeling. Am J Physiol Lung Cell Mol Physiol 2004; 287:L307-317. “ @  † ‚#   >       $ {        $  #" 

\_^  ~ $ _••_– ŒŒ’”^ŒŒ 57. Corry DB, Kiss A, Song LZ et al. Overlapping and independent contributions of MMP2 and MMP9 to lung   {     # $#   @@ #< =† > _••’– —€€^€€” — ƒ$<<  ` `  ƒ '  

\^€   %  $ ‘$      bronchopulmonary dysplasia. Am J Respir Cell Mol Biol 2009; 41:59-68. 59. Tanaka A, Yamane Y, Matsuda H. Mast cell MMP-9 production enhanced by bacterial lipopolysaccharide. J Vet Med Sci 2001; 63:811-813. 60. Fang KC, Raymond WW, Blount JL et al. Dog mast cell _-chymase activates progelatinase B by cleaving the Phe88-Phe89 and Phe91-Glu92 bonds of the catalytic domain. J Biol Chem 1997; 272:25628-25635. 61. Frank BT, Rossall JC, Caughey GH et al. Mast cell tissue inhibitor of metalloproteinase-1 is cleaved and inactivated extracellularly by _-chymase. J Immunol 2001; 166:2783-2792. 62. Kishi K, Muramatsu M, Jin D et al. The effects of chymase on MMP-2 activation in neointimal hyperplasia after balloon injury in dogs. Hypertens Res 2007; 30:77-83. 63. Ishida K, Takai S, Murano M et al. Role of chymase-dependent MMP-9 activation in mice with dextran sodium sulfate-induced colitis. J Pharmacol Exp Ther 2008; 324:422-426. 64. Inoue N, Muramatsu M, Jin D et al. Effects of chymase inhibitor on angiotensin II-induced abdominal   $  !    =^   #   _••€– _•’Œ€^Œ“’ 65. Rueff R F, Przybilla B, Bilo MB et al. Predictors of severe systemic anaphylactic reactions in patients with hymenoptera venom allergy: importance of baseline serum tryptase. J Allergy Clin Immunol 2009; 124:1047-1054. ““  # ‚\ #< \ @#  ‚@      #    !   $ lung injury. Am J Physiol Lung Cell Mol Physiol 2004; 286:L156-164. 67. Imai Y, Kuba K K, Rao S et al. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 2005; 436:112-116. 68. Reilly CF, Tewksbury DA, Schechter NB et al. Rapid conversion of angiotensin I to angiotensin II by neutrophil and mast cell proteinases. J Biol Chem 1982; 257:8619-8622. 69. Wintroub BU, Schechter NB, Lazarus GS et al. Angiotensin I conversion by human and rat chymotryptic proteinases. J Invest Derm 1984; 83:336-339. ”• '  ` #          ##   #  # ‘  II-forming enzyme in the human heart. J Biol Chem 1990; 265:22348-22357. 71. Urata H, Kinoshita A, Perez DM et al. Cloning of the gene and cDNA for human heart chymase. J Biol Chem 1991; 266:17173-17179. 72. Caughey GH, Zerweck EH, Vanderslice P. Structure, chromosomal assignment and deduced amino acid sequence of a human gene for mast cell chymase. J Biol Chem 1991; 266:12956-12963. 73. Caughey GH, Schaumberg TH, Zerweck EH et al. The human mast cell chymase gene (CMA1): mapping to the cathepsin G/granzyme gene cluster and lineage-restricted expression. Genomics 1993; 15:614-620. 74. Woodbury RG, Everitt M, Sanada Y et al. A major serine protease in rat skeletal muscle: Evidence for its mast cell origin. Proc Natl Acad Sci USA 1978; 75:5311-5313. ” &$  ‚  ~$ # `  $$     }  #      ! tissue. FEBS Lett 1980; 114:189-195. ”“ &$  ‚  $}<  ƒ$  ${$  }         small intestine. Proc Natl Acad Sci USA 1978; 75:2785-2789. ”” ## ~       % >ƒ       # ^<    #   of human mast cell. J Immunol 1990; 145:2652-2661. 78. Benyon RC, Enciso JA, Befus AD. Analysis of human skin mast cell proteins by two-dimensional gel  #            > $ €€Œ– _“€€^_”•“ 79. de Garavilla L, Greco MN, Sukumar N et al. A novel, potent dual inhibitor of the leukocyte proteases #  # $ #  ^{  !  !! > † @# 2005; 280:18001-18007. 80. Maryanoff BE, de Garavilla L, Greco MN et al. Dual inhibition of cathepsin G and chymase is effective in    $  {  > ‚ @  @   _••– —_’”^_Œ 81. Lundequist A, Tchougounova E, Abrink M et al. Cooperation between mast cell carboxypeptidase A and the chymase mouse mast cell protease 4 in the formation and degradation of angiotensin II. J Biol Chem 2004; 279:32339-32344. 82. Caughey GH, Raymond WW, Wolters PJ. Angiotensin II generation by mast cell _- and `-chymases. Biochim Biophys Acta 2000; 1480:245-257.

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

231

—Œ & @@ ` ~ $ š     #  #       ^!  } inhibitor therapy in rodents. J Clin Invest 2010. 84. Wei CC, Meng QC, Palmer R et al. Evidence for angiotensin-converting enzyme- and chymase-mediated     #   {$   #  #   !! @ $ €€€– €€_—Œ^_—€ 85. Ju H, Gros R, You X et al. Conditional and targeted overexpression of vascular chymase causes hypertension in transgenic mice. Proc Natl Acad Sci USA 2001; 98:7469-7474. 86. Shiota N, Okunishi H, Takai S et al. Tranilast suppresses vascular chymase expression and neointima formation in balloon-injured dog carotid artery. Circulation 1999; 99:1084-1090. —” <$#  *<  >       # #  ~Œ_• $  ^$ $     #  =$ > \#  _••’– ’€Œ”Œ^”“ 88. Takai S, Jin D, Muramatsu M et al. Therapeutic applications of chymase inhibitors in cardiovascular     =$ > \#  _••’– •^— 89. Mackins CJ, Kano S, Seyedi N et al. Cardiac mast cell-derived renin promotes local angiotensin formation, norepinephrine release and arrhythmias in ischemia/reperfusion. J Clin Invest 2006; 116:1063-1070. 90. Kano S, Tyler E, Salazar-Rodriguez M et al. Immediate hypersensitivity elicits renin release from cardiac mast cells. Int Arch Allergy Immunol 2008; 146:71-75. 91. K Knight PA, Wright SH, Lawrence CE et al. Delayed expulsion of the nematode Trichinella spiralis in  < # $  ^   $ # $    ^ > ="  2000; 192:1849-1856. 92. Wastling JM, Scudamore CL, Thornton EM et al. Constitutive expression of mouse mast cell protease-1 in normal Balb/c mice and its up-regulation during intestinal nematode infection. Immunology 1997; 90:308-313. 93. K Knight PA, Wright SH, Brown JK K et al. Enteric expression of the integrin _v`6 is essential for nematode-induced mucosal mast cell hyperplasia and expression of the granule chymase, mouse mast cell protease-1. Am J Pathol 2002; 161:771-779. 94. Pemberton AD, Wright SH, K Knight PA et al. Anaphylactic release of mucosal mast cell granule proteases: role of serpins in the differential clearance of mouse mast cell proteases-1 and -2. J Immunol 2006; 176:899-904. 95. Groschwitz KR, Ahrens R, Osterfeld H et al. Mast cells regulate homeostatic intestinal epithelial migration and barrier function by a chymase/Mcpt4-dependent mechanism. Proc Natl Acad Sci USA 2009; 106:22381-22386. 96. Jacob C, Yang PC, Darmoul D et al. Mast cell tryptase controls paracellular permeability of the intestine. Role of PAR-2 and beta-arrestins. J Biol Chem 2005; 280:31936-31948. 97. Mellon MB, Frank BT, Fang KC. Mast cell _^# $     #      cleaving antibody-binding epitopes. J Immunol 2002; 168:290-297. 98. Balzar S, Chu HW, Strand M et al. Relationship of small airway chymase-positive mast cells and lung function in severe asthma. Am J Respir Crit Care Med 2005; 171:431-439. 99. Waern I, Jonasson S, Hjoberg H J et al. Mouse mast cell protease 4 is the major chymase in murine airways  #   !      % { > $ _••€– —Œ“Œ“€^“Œ”“ •• ƒ} ƒ \<  $# '     #   ^ "    # mitogen-induced proliferation of human airway smooth muscle cells. J Immunol 2002; 169:1014-1020. 101. Leskinen M, Wang Y, Leszczynski D et al. Mast cell chymase induces apoptosis of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2001; 21:516-522. 102. Leskinen MJ, Heikkila HM, Speer MY et al. Mast cell chymase induces smooth muscle cell apoptosis by disrupting nff kappab-mediated survival signaling. Exp Cell Res 2006; 312:1289-1298. 103. Sun J, Zhang J, Lindholt JS et al. Critical role of mast cell chymase in mouse abdominal aortic aneurysm formation. Circulation 2009; 120:973-982. •’ ~ `\ # ‚ ! ‚ƒ  ^      $$   $  { and pathogen defense. J Biol Chem 2007; 282:20785-20789. • ! ‚ ‚ = ><#< †      $ $      =# # coli in mice. J Clin Invest 1994; 93:1645-1653. •“ ! ‚ < * ‚ =     $  $ # {$"       sites of infection through TNF_. Nature 1996; 381:77-80. 107. Sutherland RE, Olsen JS, McKinstry A et al. Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by enhancing neutrophil killing. J Immunol 2008; 181:5598-5605. 108. Huang C, Friend DS, Qiu WT et al. Induction of a selective and persistent extravasation of neutrophils into the peritoneal cavity by tryptase mouse mast cell protease 6. J Immunol 1998; 160:1910-1919. 109. Thakurdas SM, Melicoff E, Sansores-Garcia L et al. The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections. J Biol Chem 2007; 282:20809-20815.

232

MAST CELL BIOLOGY

• `$ @   * ˆ§†  \>   =!$  # $     #$   tryptase `I and demonstration of its importance in bacterial infections of the lung. J Biol Chem 2001; 276:26276-26284.     @ †$ >ƒ       #$   _-tryptase. Clin Exp Allergy 2002; 32:1000-1006. 112. Trivedi NN, Raymond WW, Caughey GH. Chimerism, point mutation and truncation dramatically transformed mast cell b-tryptases during primate evolution. J Allergy Clin Immunol 2008; 121:1262-1268. Œ * ! ~~ * } † @#$ @   `$ $‘             despite frequent inheritance of loss-off function mutations. J Allergy Clin Immunol 2009; 124:1099-1105. 114. Schwartz LB, Atkins PC, Bradford TR R et al. Release of tryptase together with histamine during the immediate cutaneous response to allergen. J Allergy Clin Immunol 1987; 80:850-855. 115. Shalit M, Schwartz LB, Golzar N et al. Release of histamine and tryptase in vivo after prolonged cutaneous challenge with allergen in humans. J Pharmacol Exp Ther 1988; 244:133-137. 116. Wenzel SE, Fowler A, Schwartz LB. Activation of pulmonary mast cells by bronchoalveolar allergen challenge. Am Rev Respir Dis 1988; 137:1002-1008. ” & $ \   < † @#    *^#  _^ ! {   ‘ $# of asthma. Am J Respir Crit Care Med 2009; 180:388-395. — ˆ# ^& \ @ ƒ ^   *  # <  % {   $ #  J Immunol 2002; 168:1992-2000. 119. Chen CL, Wang SD, Zeng ZY et al. Serine protease inhibitors nafamostat mesilate and gabexate mesilate $  ^$  % {  #   $    # >   Clin Immunol 2006; 118:105-112. 120. Mori S, Itoh Y, Shinohata R et al. Nafamostat mesilate is an extremely potent inhibitor of human tryptase. J Pharmacol Sci 2003; 92:420-423. 121. Sommerhoff CP, Sollner C, Mentele R et al. A kazal-type inhibitor of human mast cell tryptase: isolation from the medicinal leech Hirudo medicinalis, characterization and sequence analysis. Biol Chem Hoppe-Seyler 1994; 375:685-694. 122. K Krishna MT, Chauhan A, Little L et al. Inhibition of mast cell tryptase by inhaled APC366 attenuates allergen-induced late-phase airway obstruction in asthma. J Allergy Clin Immunol 2001; 107:1039-1045. 123. Erin EM, Leaker BR, Zacharasiewicz A et al. Effects of a reversible `-tryptase and trypsin inhibitor (RWJ-58643) on nasal allergic responses. Clin Exp Allergy 2006; 36:458-464. 124. He S, Aslam A, Gaca MD et al. Inhibitors of tryptase as mast cell-stabilizing agents in the human airways: effects of tryptase and other agonists of PAR-2 on histamine release. J Pharmacol Exp Ther 2004; 309:119-126. 125. He S, Gaca MD, Walls AF. A role for tryptase in the activation of human mast cells: modulation of histamine release by tryptase and inhibitors of tryptase. J Pharmacol Exp Ther 1998; 286:289-297. 126. Molinari JF, Moore WR, Clark J et al. Role of tryptase in immediate cutaneous responses in allergic sheep. J Appl Physiol 1995; 79:1966-1970. 127. Clark JM, Abraham WM, Fishman CE et al. Tryptase inhibitors block allergen-induced airway and {      #  > ‚ @  @   €€– __•”“^_•—Œ 128. Wright CD, Havill AM, Middleton SC et al. Inhibition of allergen-induced pulmonary responses by the selective tryptase inhibitor 1,5-bis-[4-[(3-carbamimidoyl-benzenesulfonylamino)- methyl]-phenoxy]-pen tane (amg-126737). Biochem Pharmacol 1999; 58:1989-1996. 129. Rice KD, Wang VR, Gangloff AR R et al. Dibasic inhibitors of human mast cell tryptase. Part 2:Structureactivity relationships and requirements for potent activity. Bioorg Med Chem Lett 2000; 10:2361-2366. 130. Cairns JA. Inhibitors of mast cell tryptase `  # $  #    #  {  disorders. Pulm Pharmacol Ther 2005; 18:55-66. Œ ‚$ > `  * @$# `         $$    > @ ! 1991; 88:493-499. 132. Hartmann T, R Ruoss SJ, Caughey GH. Modulation of thrombin and thrombin receptor peptide mitogenicity by human lung mast cell tryptase. Am J Physiol Lung Cell Mol Physiol 1994; 267:L113-L119. ŒŒ $ †ƒ % ‚‚ ><    `$   !          $   $  #    #" > $ €€”– 158:2310-2317. Œ’ @  > &      $ #      #$ $   J Clin Invest 1997; 99:1313-1321. 135. Brown JK, Tyler CL, Jones CA et al. Tryptase, the dominant secretory granular protein in human mast cells, is a potent mitogen for cultured dog tracheal smooth muscle cells. Am J Resp Cell Mol Biol 1995; 13:227-236. 136. Blair RJ, Meng H, Marchese MJ et al. Human mast cells stimulate vascular tube formation. Tryptase is a novel, potent angiogenic factor. J Clin Invest 1997; 99:2691-2700.

MAST CELL PROTEASES AS PROTECTIVE AND INFLAMMATORY MEDIATORS

233

137. McNeil HP, Shin K, Campbell IK K et al. The mouse mast cell-restricted tetramer-forming tryptases mMCP6   @\”        {   #   #  ‚#$ _••—– —_ŒŒ—^_Œ’“ Œ— #  ~ ! \ @ # >      $  $$ {   #  ! # tryptase/heparin complexes. J Immunol 2009; 182:647-656. 139. Buckley MG, Walters C, Wong WM et al. Mast cell activation in arthritis: detection of _- and `-tryptase, #  #     ! {$ @  ˜@#™ €€”– €ŒŒ“Œ^Œ”• ’• ƒ      $ #       $ < % $  {  arthritis. Science 2002; 297:1689-1692. 141. Nigrovic PA, Binstadt BA, Monach PA et al. Mast cells contribute to initiation of autoantibody-mediated arthritis via IL-1. Proc Natl Acad Sci USA 2007; 104:2325-2330. 142. Nigrovic PA, Lee DM. Synovial mast cells: role in acute and chronic arthritis. Immunol Rev 2007; 217:19-37. ’Œ  #$ >  &           W-sh mice does not impair antibody-mediated arthritis. J Exp Med 2007; 204:2797-2802 144. Balázs M, Illyés G, Vadász G. Mast cells in ulcerative colitis. Quantitative and ultrastructural studies. Virchows Arch 1989; 57:353-360. 145. Fox CC, Lazenby AJ, Moore WC et al. Enhancement of human intestinal mast cell mediator release in active ulcerative colitis. Gastroenterology 1990; 99:119-124. 146. Tremaine WJ, Brzezinski A, Katz JA et al. Treatment of mildly to moderately active ulcerative colitis with a tryptase inhibitor (APC2059): an open-label pilot study. Aliment Pharmacol Ther 2002; 16:407-413. 147. Isozaki Y, Yoshida N, K Kuroda M et al. Anti-tryptase treatment using nafamostat mesilate has a therapeutic effect on experimental colitis. Scand J Gastroenterol 2006; 41:944-953. 148. R Rubinstein I, Nadel JA, Graf PD et al. Mast cell chymase potentiates histamine-induced wheal formation in the skin of ragweed-allergic dogs. J Clin Invest 1990; 86:555-559. 149. Briggaman RA, Schechter NM, Fraki J et al. Degradation of the epidermal-dermal junction by proteolytic enzymes from human skin and human polymorphonuclear leukocytes. J Exp Med 1984; 160:1027-1042. 150. Sommerhoff CP, Caughey GH, Finkbeiner WE et al. Mast cell chymase. A potent secretagogue for airway gland serous cells. J Immunol 1989; 142:2450-2456. 151. Sommerhoff CP, Nadel JA, Basbaum CB et al. Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells. J Clin Invest 1990; 85:682-689. 152. Matin R, Tam EK, Nadel JA et al. Distribution of chymase-containing mast cells in human bronchi. J Histochem Cytochem 1992; 40:781-786. 153. Raymond WW, Waugh R Ruggles S, Craik CS et al. Albumin is a substrate of human chymase: prediction by combinatorial peptide screening and development of a selective inhibitor based on the albumin cleavage site. J Biol Chem 2003; 278:34517-34524. 154. Muramatsu M, Katada J, Hattori M et al. Chymase mediates mast cell-induced angiogenesis in hamster sponge granulomas. Eur J Pharmacol 2000; 402:181-191. 155. Abonia JP, Friend DS, Austen WG Jr et al. Mast cell protease 5 mediates ischemia-reperfusion injury of mouse skeletal muscle. J Immunol 2005; 174:7285-7291. 156. Sun J, Sukhova GK, Yang M et al. Mast cells modulate the pathogenesis of elastase-induced abdominal aortic aneurysms in mice. J Clin Invest 2007; 117:3359-3368. 157. Shiota N, Fukamizu A, Takai S et al. Activation of angiotensin II-forming chymase in the cardiomyopathic hamster heart. J Hypertens 1997; 15:431-440. 158. Takai S, Shiota N, Kobayashi S et al. Induction of chymase that forms angiotensin II in the monkey atherosclerotic aorta. FEBS Lett 1997; 412:86-90. 159. Takai S, Yuda A, Jin D et al. Inhibition of chymase reduces vascular proliferation in dog grafted veins. FEBS Lett 2000; 467:141-144. “• ~#  *<         #^  ^  #% in the development of vascular proliferation. Circulation 2001; 104:1274-1279. 161. Wolters PJ, Laig-Webster M, Caughey GH. Dipeptidyl peptidase I cleaves matrix-associated proteins and is expressed mainly by mast cells in normal dog airways. Am J Respir Cell Mol Biol 2000; 22:183-190. 162. Wolters PJ, Raymond WW, Blount JL et al. Regulated expression, processing and secretion of dog mast cell dipeptidyl peptidase I. J Biol Chem 1998; 273:15514-15520. 163. Turk D, Janjic V, Stern I et al. Structure of human dipeptidyl peptidase I (cathepsin C): exclusion domain added to an endopeptidase framework creates the machine for activation of granular serine proteases. EMBO J 2001; 20:6570-6582. 164. McGuire MJ, Lipsky PE, Thiele DL. Generation of active myeloid and lymphoid granule serine proteases requires processing by the granule thiol protease dipeptidyl peptidase I. J Biol Chem 1993; 268:2458-2467. 165. Pham CT, Ley TJ. Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo. Proc Natl Acad Sci USA 1999; 96:8627-8632. 166. Wolters PJ, Pham CT, Muilenburg DJ et al. Dipeptidyl peptidase I is essential for activation of mast cell chymases, but not tryptases, in mice. J Biol Chem 2001; 276:18551-18556.

234

MAST CELL BIOLOGY

167. Caughey GH. A pulmonary perspective on GASPIDs: Granule-associated serine peptidases of immune defense. Curr Resp Med Rev 2006; 2:263-277. 168. Adkison AM, Raptis SZ, Kelley DG et al. Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis. J Clin Invest 2002; 109:363-371. 169. Pham CT, Ivanovich JL, Raptis SZ et al. Papillon-Lefevre syndrome: correlating the molecular, cellular   ‹$  # @      #$ > $ _••’– 173:7277-7281. 170. Vanderslice P, Ballinger SM, Tam EK K et al. Human mast cell tryptase: multiple cDNAs and genes reveal a multigene serine protease family. Proc Natl Acad Sci USA 1990; 87:3811-3815. 171. Vanderslice P, Craik CS, Nadel JA et al. Molecular cloning of dog mast cell tryptase and a related protease: structural evidence of a unique mode of serine protease activation. Biochemistry 1989; 28:4148-4155. 172. Sakai K, Ren S, Schwartz LB. A novel heparin-dependent processing pathway for human tryptase: autocatalysis followed by activation with dipeptidyl peptidase I. J Clin Invest 1996; 97:988-995. 173. Henningsson F, Yamamoto K, Saftig P et al. A role for cathepsin E in the processing of mast-cell carboxypeptidase A. J Cell Sci 2005; 118:2035-2042. 174. Henningsson F, Wolters P, Chapman HA et al. Mast cell cathepsins C and S control levels of carboxypeptidase A and the chymase, mouse mast cell protease 5. Biol Chem 2003; 384:1527-1531.

CHAPTER R 13 MAST CELLS IN LUNG INFLAMMATION Elena P. Moiseeva1  \ † |,1,2 1

{  < j < | j ^ 

j ' | j  < > Z| 8 ^ < >| >| 8#~ {  < `^   |  

Z| >| 8#    _ 0

  &

 

2

Abstract:

Mast cells play an important role in the lung in both health and disease. Their                {    response to tissue damage initiated by a variety of diverse stimuli. They are important for host immunity against bacterial infection and potentially in the host immune response to non small cell lung cancer. In situations of ongoing tissue damage, the $   $ $  ^{       < contributes to the pathophysiology of lung diseases such as asthma and interstitial lung disease. A key goal is the development of treatments which attenuate adverse mast cell function when administered chronically to humans in vivo. Such therapies may offer a novel approach to the treatment of many life-threatening diseases.

INTRODUCTION In this chapter, we will examine the role of mast cells (MCs) in the initiation and    %   # {    @     role in host defense against pathogens, particularly bacterial infection and in tissue repair # $# # #    $ $  ^{       cytokines, the pattern of which varies depending on the stimulus. In healthy human lung, MCs are ubiquitous in the airways and parenchyma, but redistribute to key tissue structures in disease. Their location at the interface between the external environment and lung tissue places them in the ideal location to respond rapidly to perceived tissue insults. MCs can respond to many diverse stimuli (Fig. 1) and it is our view that their primary role is to initiate         {      $  $ #  # $$  #  # $ $    $ `$ $  cells (HLMCs) achieve their biological effects through cell-cell interactions and the release Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 235

236

MAST CELL BIOLOGY

Figure 1. Potential mechanisms of human lung mast cell activation.

of numerous autacoid mediators (histamine, prostaglandin [PG]D2, leukotriene [LT]C4), proteases and multifunctional cytokines and chemokines. The biological activities of these mediators are summarized in Tables 1-3. The release of these mediators is not all-or-none, $ ## $  $$^  *# % # @    %#  $$  cells, as well as cells of both the innate and acquired immune systems.

MAST CELL RECRUITMENT TO THE LUNG The mechanisms of MC homing to human lung are poorly understood and the primary signals are unknown. However, MC expansion in sensitized murine lung is induced by Type 2 NKT cells and IL-9 after allergen challenge and PGE2, produced by nasal mucosa.1,2 In addition, systemically available sphingosine 1-phosphate (S1P) increases

MAST CELLS IN LUNG INFLAMMATION

237

Table 1. The classical human mast cell mediators and their biological effects Mediator

Activity

Histamine (stored)

Bronchoconstriction; tissue edema; Bvascular permeability; B mucus secretion; B     – Bcollagen synthesis; B endothelial cell proliferation, dendritic cell activation

Heparin (stored)

Anticoagulant; storage matrix for mediators; sequestering of growth      –   !– #  migration

Tryptase T (stored)

Degrades respiratory allergens and cross-linked IgE; generates C3a and bradykinin; degrades neuropeptides; increases BHR R and ASM contractility; indirectly activates collagenase; B        #– # ! potentiates mast cell histamine release; neutrophil recruitment; TGF-beta activation

Chymase (stored)

B mucus secretion; ECM degradation, T Type I procollagen processing, angiotensin II synthesis, ? T-cell adhesion to airway smooth muscle,

activates IL-1b, degrades IL-4, releases membrane-bound SCF PGD2 (synthesized)

Bronchoconstriction; tissue edema; B mucus secretion; dendritic cell activation; chemotaxis of eosinophils, Th2 T cells and basophils via the CRTH2 receptor

LTC4/D4 (synthesized)

Bronchoconstriction; tissue edema; B mucus secretion; enhances IL-13-dependent airway smooth muscle proliferation; dendritic cell maturation and recruitment; eosinophil IL-4 secretion, mast cell homing, mast cell IL-5, IL-8 and TNF_  – $  

@ $   #! ! ˜†ƒ™ {$  3 Mediators from mature MCs may also recruit further immature MC progenitors4 (see Tables 1 and 2). In addition, the $ @ $  "  #  $#    ! !  stimuli, including stem cell factor (SCF), S1P and other growth factors. There is evidence that several factors may lead to the redistribution of MCs in human lung. For example, HLMCs express CXCR3, particularly on the cells within the ASM bundles in asthma, which in turn preferentially express the chemotactic ligand CXCL10.5

MAST CELLS IN HEALTHY LUNG HLMCs show a marked heterogeneity within the tissue with variability in their size and shape, protease and cytokine content and response to various stimuli (reviewed in ref. 6). In comparison with MCs from other sources, HLMCs display distinct differences in #  $  < "    #<      released mediators.7-12 HLMCs belong to either a predominant tryptase (MCT) or tryptase/chymase

(MCTC) subtype. The rare chymase-only (MCC™ $ #     %# # lung in several studies.7,9,13 Isolated lung MCT are smaller and produce lower amounts of the autacoid mediators histamine and PGD2 compared with MCTC cells.10,14,15 In situ,

238

MAST CELL BIOLOGY

Table 2. Biological activity of human mast cell-derived chemokines in vitro Chemokine

T Target Cells

Biological Effects

CCL1

T cells

T-cell recruitment

CCL2

Mast cells Epithelial cells Fibrocytes T cells Eosinophils Monocytes Basophils

Chemotaxis Proliferation, chemotaxis Chemotaxis ¶*#_ # Chemotaxis Chemotaxis Activation, mediator release

CCL3

Mast cells T cells Macrophages Neutrophils Eosinophils Monocytes Basophils

Activation, mediator release ¶*# #  #" ˜*#^!™ Differentiation Chemotaxis (in vivo) and cytotoxicity Chemotaxis Chemotaxis Activation, mediator release

CCL4

T cells Eosinophils Neutrophils

¶*# #  #" ˜*#^!™ Chemotaxis Chemotaxis (in vivo)

CCL5

Mast cells T cells Eosinophils Monocytes

Chemotaxis? ¶*# #  #" ˜*#^!™ Chemotaxis Chemotaxis

CCL7 L

Eosinophils Monocytes Basophils

Chemotaxis Chemotaxis Activation, mediator release

CCL12

Fibrocytes Monocytes Eosinophils L Lymphocytes

Chemotaxis Chemotaxis Chemotaxis Chemotaxis

CCL17

T cells

Chemotaxis (Th2-selective)

CCL19

Airway smooth muscle

Chemotaxis

CCL20

Dendritic cells T cells

Chemotaxis Chemotaxis

CCL22

T cells

Chemotaxis (Th2-selective)

CXCL5

Neutrophils

Chemotaxis

CXCL8

Mast cells Endothelial cells Neutrophils Eosinophils

? chemotaxis, ? mediator release

Proliferation, survival, chemotaxis, angiogenesis Chemotaxis Chemotaxis after priming with IL-3, IL-5 or GM-CSF

MAST CELLS IN LUNG INFLAMMATION

239

Table 3. Biological activity of human mast cell-derived cytokines in vitro Cytokine IL-4

T Target Cells B cells T cells Monocyte/macrophages

Eosinophils Fibroblasts Endothelial cells Mast cells

Biological Effects proliferation; B MHC class II, B CD40, B CD25; B IgE, B IL-6 Proliferation, ATh2 cell phenotype ? H 2O2 and O2–, ? parasite killing, ? $  !–  ¶  phage differentiation; ? IL-1, IL-6, IL-8 and TNF B MHC class II and CD23; 15-lipoxygenase expression Transendothelial migration T Proliferation, chemotaxis, B ECM; B ICAM-1 Proliferation, B VCAM-1, ? ICAM-1 B Fc¡RI, B ICAM-1

IL-3, IL-5, GM-CSF

Eosinophils

Growth, adhesion, transendothelial migration, chemotaxis, activation and prolonged survival

IL-6

B cells T cells Mast cells

Ig secretion including B IgE Growth, differentiation Survival

IL-13

B cells Monocyte/macrophages Eosinophils Endothelial cells

IgE synthesis As for IL-4 Activation, survival B VCAM-1

TNF_

Monocyte/macrophages

B cytotoxicity, chemotaxis, prolonged survival

T cells Neutrophils Eosinophils Endothelial cells Fibroblasts

Class II antigen and IL-2R R expression, proliferation B cytotoxicity, chemotaxis B cytotoxicity, oxidant production B E-selectin, B ICAM-1, B VCAM-1; B intravasation of most leucocytes Growth, chemotaxis, ? collagen, Bcollagenase, B IL-6, B IL-8 Histamine and tryptase secretion

Mast cells SCF

Mast cells

Growth, differentiation, survival, chemotaxis

NGF

B cells T cells Eosinophils Basophils Neutrophils Monocyte/macrophages Fibroblasts Smooth muscle cells

Differentiation, proliferation, B Ig Differentiation, proliferation Proliferation Activation, mediator release Chemotaxis, survival, mediator release Proliferation, mediator release Migration, contraction Migration, contraction, proliferation continued on next page

240

MAST CELL BIOLOGY

Table 3. Continued Cytokine

T Target Cells

Biological Effects

Mast cells

Differentiation, survival, activation, mediator release

TGF`

Smooth muscle cells Epithelial cells Endothelial cells

Differentiation, activation Inhibition of proliferation B angiogenesis

FGF-2

Fibroblasts Endothelial cells

Proliferation B angiogenesis

IFN_

NK K cells Macrophages Dendritic cells T cells

B cytotoxicity

Dendritic cells Mast cells

induction of Th2 immunity BIL-13 expression

TSLP

Development, maturation Activation, maturation, B IFNγ B survival of activated T cells, B Th1 phenotype

the size of lung MCT and MCTC cells varies according to the location.16 In addition, MCTC cells accumulate other peptidases, express CD88 and a distinct set of cytokines and chemokine receptors.8-10,17,18 Their properties are summarized in Table 4. The MCT phenotype is typically found in the lung parenchyma, bronchial epithelium and the bronchial lamina propria, whereas the MCTC phenotype localizes around blood vessel walls.8,9,16,19 Andersson     !  ^  $ %# ! @T and MCTC types on the basis of expression patterns for the IgE receptor, IL-9 receptor, renin, histidine decarboxylase, VEGF, basic FGF (bFGF, FGF2), 5-lipoxygenase and leukotriene C4 synthase. Interestingly, renin expression is observed predominantly in the MCT subtype and only in pulmonary vascular MCTC cells. The relatively high expression  # ##^  =   #  "       % MCT and MCTC cells, with IgE receptor virtually absent in alveolar MCs, are likely to be of particular physiological importance. A differential expression pattern of proteases among MCs and the varying anatomical location of these cells indicate that they will have distinct roles in normal lung physiology and disease, but these remain poorly understood. MCs subtypes develop in the presence of      #%  ##    ¢  !   $$  @TC cells to MCT has been reported in coculture with human airway epithelium.20 Hence, it is perhaps not surprising that MCs adopt the MCT subtype in close proximity to epithelial cells in the alveoli and bronchi. In contrast, coculture of MCT cells with endothelial cells transforms them into the MCTC subtype,21 which may explain the predominance of the MCTC subtype around blood vessels. *< #  # "  ^  $$  `ƒ @  <   a response to local environmental cues affecting gene expression, resulting in appropriate physiological function.16 For example, renin expression in combination with chymase, converting AngI into AngII, in MCTC cells close to pulmonary vasculature, as well as VEGF expression in MCs around pulmonary vessels, suggests involvement of these MCs in local regulation of vascular function.

Responsiveness to stimuli

Cytokines, chemokines and receptors

Protease content   biomarkers

Parameter Location and percentage

Mast Cells

MCT, MCTC and rare MCC. MC density is increased in small airways and alveolar parenchyma compared with central airways.16 Decreased renin in central airways compared to other locations.16 Increased FcepsilonRI and histidine decarboxylase, decreased 5-lipoxygenase in central airways compared to other locations.16 @@‚Œ  @@‚  «œ  @– @@‚Œ  @@‚’  «Œ•œ @   ƒ€‚   airways and around pulmonary vessels compared to central airways; increased VEGF around pulmonary vessels.16

7

Total Population T

FcepsilonRI, substance P.10

IL-4(low), IL-5, Il-6, IL-13 (low).9

Normal Lung 85-95% in the alveolar walls, bronchioles and bronchi.8,9,16,19 8 T Tryptase.

MCT Subtype

continued on next page

FcepsilonRI, C5a, compound 48/80, substance P.10

IL-4(high), IL-13 (high),9 CCR3.18

CD88,10 increased leukotriene C4 synthase in small airways and around blood vessels.16

4-15% in alveolar attachments, small airway outer wall and in bronchi,8,9,16,19 «•œ  $ pulmonary vessels.16 cathepsin G,17 carboxypeptidase A.8

MCTC Subtype

Table 4. Phenotypes and localization of lung mast cells in normal lung and in disease

MAST CELLS IN LUNG INFLAMMATION 241

Increased amphiregulin103 and thymic stromal lymphopoietin.104

  biomarkers

continued on next page

100% IL-4-positive vs 60% in control.9

7-fold increased TNF-alpha expression.88 55% of MCs within the ASM express IL-4 and 17% express IL-13.101 Increased MC number correlates with CCR1 and CCR4 R -expression in asthma; CCR1 and CCR4 R in 24% and 19% of MCs, respectively.102 CXCR3 is expressed in 100% of MCs in ASM and 47% of MCs in the submucosa.5

Cytokines, chemokines and receptors

MCTC Subtype

\  $    

(83%).98 Higher numbers and the MCTC/ MCT ratio in alveolar attachments in severe asthma.13 Increased density around blood vessels in the lamina propria.99 Increased density in the adventitial layer of small pulmonary arteries in fatal asthma.100

Asthma

MCT Subtype

Increased density and degranulation in epithelium,87,88,91,93 in lamina propria,93 the airway mucous glands,96,97 ASM.96-98 Increased percentage in small airway outer and inner wall regions, alveolar attachments, peripheral alveolar tissue and large airway in severe asthma.13 Increased numbers around small blood vessels in bronchi.99 Increased density in the adventitial layer of small pulmonary arteries in fatal asthma.100

Total Population T

Location

Mast Cells

Table 4. Continued

242 MAST CELL BIOLOGY

  biomarkers

Reduced net MC density with increased luminal MC number.107 Reduced density in submucosa and ASM bundle, but increased in glands (did not reach   ™108

Increased expression of CD88.107

continued on next page

Increased expression of CD88 and TGF-beta and decreased rennin.107

pulmonary vessels and alveolar parenchyma in very severe COPD.107

subepithelial area of airways vs controls.107

*#  %    !    % # $    #  # ASM and the PC 20 for methacholine in the subjects with asthma.98 The MCTC number and the MCTC/MCT ratio in the small airway outer wall and alveolar attachments positively correlate with lung function in severe asthma.13 The density in the lamina propria is related to the vascular area and to the number of VEGF+ MC in mild to moderate asthma,99 with the MCTC/MC ratio correlated with the PD20 methacholine values.99

MCTC Subtype

Higher numbers in both subepithelial area and ASM area of peripheral airways, but not in controls.109 Increased density in walls of small airways and in alveolar parenchyma vs control;107 several-fold increase in small airways, small airway epithelium,

MCT Subtype

Lower density in the subepithelial area of central airways vs. controls.109 Lower numbers in the

COPD

MC localization to the bronchial ASM is associated with airway hyperresponsiveness in asthma.105 The numbers of total MCs and degranulated MCs correlate with the lumen area occupied by mucus.106

Functional correlations

Location

Total Population T

Mast Cells

Table 4. Continued

MAST CELLS IN LUNG INFLAMMATION 243

MCT Subtype

increased bFGF in lung interstitium, surrounded           cells.114-116

@ $     %# #     110

  biomarkers

Functional correlations

Fibrotic Lung Disorders

Cell numbers in the epithelium and subepithelial area of peripheral airways positively correlate with FEV1/VC lung function, the cell percentage in the total airway wall area of peripheral airways positively correlate with higher values of FEV1 and FEV1/ VC.109

COPD (continued)

Increased MC number in the alveolar septa and within alveoli.110 Increased MC density and activation in bronchi.111-113

Total Population T

Location

Functional correlations

Mast Cells

Table 4. Continued

MCTC numbers correlate with accumulation        proliferation.113

Increased MCTC counts in ASM cells.113

Cell numbers in ASM of peripheral airways positively correlate with FEV1% pred lung function. Higher numbers in the adventitia of peripheral airways tend to correlate positively with FEV1% pred.109

MCTC Subtype

244 MAST CELL BIOLOGY

MAST CELLS IN LUNG INFLAMMATION

245

MAST CELLS IN LUNG DEFENSE Since the role of MCs in the immune response is addressed in other chapters, here we present only those data which are directly related to respiratory infection and lung { Defense against Bacterial Infections Experimental animal models suggest that MCs are indispensible for the clearing of pulmonary infections caused by respiratory pathogens such as Klebsiella pneumoniae, Mycoplasma pneumoniae, Streptococcus pneumoniae and Mycobacterium tuberculosis. Direct contact of MCs with clinical isolates of K. pneumoniae Cllll decreased bacterial viability after 60 min in vitro, indicating involvement of MCs in the innate immune response.22   $ $   ^  W/Wv mice had a much higher burden of K. pneumoniae KP1415 in the lungs compared with wild type or KitW/Wv mice reconstituted with MCs.23   @^  W-sh/KitW-sh mice infected with K. pneumoniae intranasally have 4-fold worse survival after 4 days compared to wild-type mice.24 KitW-sh/KitW-sh mice are more likely to die from M. pneumoniae compared with wild-type mice.25 *#  $      @^   with a 162-fold difference 4 days following infection when compared to wild-type mice. At later time points, bacterial burden is similar in both mouse strains, but neutrophilic {  !   @^  25 This latter observation raises the  # @   #!  ^{    # "  ! bacterial infection. MCs have the ability to bind and engulf bacteria and they also release a number of {    $ *~_, IL-4, IL-6, IL-10 and leukotrienes in response to bacterial infection. Exposure of the rat MC line RBL-2H3 to M. pneumoniae induces production of mRN R A for IL-4, IL-6 and TNF_ within 2 hours.26 Interestingly, expression of the Fc¡RI alpha chain expression is required for activation of MCs for the IL-4 production induced by M. pneumoniae.27 Several MC mediators are crucial for a full antibacterial response against acute infection. TNF_ released locally by MCs plays a central role by recruiting neutrophils to sites of infection. TNF_ is induced in response to bacterial FimH binding to the CD48 receptor in MCs and contributes to host defense against K. pneumoniae.23 Inhibition of TNF_ activity within the lung results in markedly decreased both short- and long-term survival of mice challenged with K. pneumoniae intratracheally.28 Likewise, susceptibility to pneumonia in some strains of mice, infected with S. pneumoniae, correlates with the number of MCs and the speed of TNF_ release in the lung, which is required for recruitment of circulating leukocytes.29 @^  W-sh/KitW-sh mice selectively reconstituted with IL-6
246

MAST CELL BIOLOGY

$ #   @     ‹$   #    # @^  proteases. For example, mice lacking protease-6, similar to human tryptase beta 1, cannot clear K. pneumoniae      $31 These mice have 8-fold lower survival after 3 days compared with that of wild-type mice. Increased lethality in these mice is associated with higher bacterial burden and lower neutrophil recruitment despite normal MC production of IL-6 and TNF_ ƒ<% %# @^  W/W(v) mice were given enzymatically active human tryptase beta I or its inactive zymogen before pulmonary infection with K. pneumoniae, tryptase beta I-treated mice had ¾5 fold fewer viable bacteria in their lungs 6 hours after inoculation relative to zymogen-treated mice.32 When enzymatically active tryptase beta I, but not tryptase alpha, was instilled into the lungs of mice, neutrophil numbers in BAL increased 100-fold, but methacholine-induced bronchoconstriction was not altered. These data suggest that mMCP-6/tryptase beta I is the primary preformed granule mediator that protects mice from acute bacterial infections. The mechanism of neutrophil recruitment by mMCP-6/tryptase beta I is not clear, but the experimental data suggest that it is not due to IL-8, IL-6 or TNF_,31,32 but may involve neutrophil chemotactic factors, produced by other cell types stimulated by tryptase.33 In summary, experimental models of acute respiratory bacterial infection show that MCs are critical in the early stages of infection and have a profound effect on survival for days after the onset of infection. Mouse models show that MCs contribute to innate defenses against respiratory pathogens. Defense against Viral Infection Respiratory viral infections cause accumulation of MCs, as well as T and B lymphocytes $  #      $  ‹$    #  $34 ="$   #$ @     ! $ $#  {$} ! $ ˜\‚—™ *  reovirus and respiratory syncytial virus (RSV), induced substantial production of IFN_; a similar response was observed upon stimulation of these cells with the TLR3 ligand, poly I:C.35 Poly I:C-induced IFN_ production involved activation of NF-kappaB, p38 and C-Jun NH2-terminal kinase and mitogen-activated protein kinase. Activation of TLR3 with poly I:C did not induce TNF_, IL-1`, IL-5, or GM-CSF production, in contrast to activation  *ƒ‚^_ ’   €   ƒ\ {  @  !35 In addition, treatment with dsRN R A alone or with IgE did not lead to MC degranulation.35 Similarly, degranulation does not occur by direct RSV infection into MCs, however, it was detected  @ } %#   =36,37 Furthermore, coculture with RSV-infected lung cancer A549 cells with the MC line HMC-1 induced degranulation in HMC-1 cells which coincided with upregulation of TNF_ secretion. In this case, degranulation is likely to be mediated by defensins released from infected epithelial cells.38 These data suggest that MC degranulation may occur during respiratory viral infection in vivo. In keeping with this, RSV infection increases the release of leukotrienes from MCs in rat airways.39  %#  ! $ $#  ‚ž  {$} ! $ $ ! $^  IgE in humans and experimental models.37,40,41 =       ! $  implicated in MC degranulation, resulting in asthmatic and allergic responses in mouse models of allergic diseases.41,42 Similar to viral infections, respiratory bacterial infections with Chlamydia pneumoniae, Z  ' [, S. pneumoniae and M. pneumoniae     $    =     $  43-45 Moreover, the  !   ^  =    # #  #   #44,45 It is therefore plausible that on reinfection, a simultaneous engagement of TLRs, other

MAST CELLS IN LUNG INFLAMMATION

247

 ^     ¡RI causes a synergistic activation of MCs, further     ^{     $ $   $ $ *#   @   <  !!  # "   $  during viral and bacterial infections and leading to deterioration of airway physiology.

  " 8; ‚   &@> * The functional response of HLMCs to bacterial, viral and other ligands has not been $   `ƒ @ #% ! *ƒ‚ "       %# @ from umbilical cord, peripheral blood or skin.35,46,47 They express relatively high levels of TLR-2, -3, -4 and -7, lower levels of TLR-1, -5 and -10, whereas expression of TLR-6, -8, or -9 is not observed in these cells.47 In contrast to HLMCs, murine peribronchial MCs strongly express TLR-9.48 Exposure of HLMCs to the TLR2 ligand, lipoteichoic acid, reduced both Fc¡RI expression and IgE-induced degranulation, but the TLR2/TLR6 ligand, peptidoglycan, did not have similar effects.49 These data suggest that some bacterial TLR2 ligands may modify some aspects of allergic disease. In murine lung MCs, a synthetic ligand for TLR2/TLR1 heterodimers, tripalmitoyl Cys-Ser-(Lys)4, induced JE (murine equivalent to human chemokine CCL2) production via NF-kappaB activation, but did not induce Ca2 mobilization or degranulation.50 These responses observed in murine cells were in      @2 mobilization and degranulation in the human LAD-2 MC line, stimulated with the same TLR2 agonist.50 TLR2
MAST CELLS IN CHRONIC LUNG DISEASE # Asthma is an airway disease that is characterized by airway obstruction due to  #  $$ #    % {    remodeling of various airway elements. Chronic MC activation and the relocation of #  %#    %  $$    <   # #   @ !  $  # #  {  $  (reviewed in ref. 52).

    ;;  # There is compelling evidence, based largely on studies from humans, that MCs play  !   # #  # *#    #   ^{  mediators which are directly relevant to the pathophysiology of asthma (see Tables 1-3). For example, the autacoid mediators, histamine, PGD2 and LTC4, induce bronchoconstriction, mucus secretion and mucosal edema and thus contribute to the symptoms of asthma.

@  #}     $   {  < ˜$ ƒ^’

248

MAST CELL BIOLOGY

IL-5 and IL-13), which regulate both IgE synthesis and the development of eosinophilic {–  !      < $ * `and bFGF. Moreover, the serine proteases tryptase and chymase, which are major secretory products of human MCs, can interact with various cell types and profoundly alter their behavior.

   =# $]# @+ # Approximately 90% of subjects with asthma under the age of 30 are atopic, with positive skin prick tests to common aeroallergens such as the house dust mite (reviewed in see ref. 53). The role allergen exposure plays in day-to-day chronic asthma is probably over-stated, but the ability of pollen to exacerbate asthma,54,55 and the ability of anti-IgE therapy to attenuate the disease in some patients indicates that allergy remains a relevant factor in asthma pathophysiology. Bearing this in mind, the technique of acute bronchial challenge with a relatively large dose of allergen in the laboratory has provided a useful model for studying asthma pathophysiology. Following bronchial allergen challenge, most atopic asthmatics and many atopic nonasthmatics experience a rapid fall in pulmonary function (e.g., forced expiratory volume in one second [FEV1]) which peaks at 10-20 $  #  $ !  ! # % _ #$  *#     # early asthmatic response (EAR). In about 50% of subjects, after 4-6 hours there is a further fall in FEV1, termed the late asthmatic response (LAR). This may last up to 12 hours and in some individuals may be followed by recurring airway obstruction for several days or even weeks (reviewed in see ref. 53). & $*: Several vasoactive and spasmogenic mediators are released during the EAR, most of which originate from MCs resident in the airway mucosa. The relative rate of mediator release from HLMCS in vitro is histaminePGD2LTC4 with one-half maximal release occurring at 2, 5 and 10 minutes respectively.56 *#  {  !!  # !   #    †ƒ {$ %# ^• $ %  bronchial allergen challenge.57-63 It has been calculated that the concentrations of mediators generated are similar to those required to produce bronchoconstriction in vitro62 or when delivered by nebuliser.60 Evidence for their role is cemented by studies using potent and !   %##   # # =‚    $ by antagonists of histamine, LTC4 and to a lesser extent PGD2.64-68 The MC as a source of these mediators is supported by four lines of evidence. Firstly, # <  =^    !!   #  $   @  !   #    @ ! $  # =‚     #        #    @^      !   BAL within minutes following local bronchial allergen challenge.58,63 Thirdly, `-agonists such as salbutamol, known inhibitors of MC degranulation when applied acutely in vitro, completely abolish the early reaction and the associated increase in plasma histamine levels.69-71 Lastly, the EAR R is markedly attenuated after pretreatment with anti-IgE,72,73   # =^     & *: In contrast to the acute mediator-induced bronchoconstriction and mucosal edema characteristic of the EAR, the LAR R is associated with the recruitment and activation of many cell types including eosinophils, activated CD4 T cells and activated macrophages.74-79 Subsequent mediator release and tissue damage following these events is thought to account for the ensuing airway obstruction and associated increase in bronchial hyperresponsiveness (BHR) which accompanies the LAR. The role of the MC   $    #   $  # ƒ‚     $    #

MAST CELLS IN LUNG INFLAMMATION

249

during the EAR R due to the recruitment and activation of many cell types, but the LAR R is also attenuated markedly by anti-IgE therapy,72 indicating that MC activation during the EAR R initiates events leading to the LAR. It is therefore likely that the secretion of MC mediators and cytokines orchestrate the development of the LAR.

   ; "  # !; Of particular relevance to chronic everyday asthma, MCs in asthmatic airways exhibit features of chronic on-going activation (summarized in Table 4). There are increased $   @  †ƒ {$    #       57,80,81 together with increased concentrations of histamine and tryptase, providing evidence of on-going MC degranulation.57,58,82 MCs from symptomatic asthmatic subjects exhibit greater constitutive histamine release in BAL and the secretory response of asthmatic BAL MCs to IgE-dependent activation is altered, with reports of increased IgE-dependent histamine release from atopic asthmatic BAL MCs compared to non-atopic normal controls.81,82 Strong correlations have been observed between the severity of BHR R and MC numbers, #    $ #   †ƒ {$57,81,83,84 There is morphological evidence of continuous degranulation as assessed by microscopy in the airway epithelium, submucosa and smooth muscle in asthma.85-87 Finally, there is increased protein expression and transcription for the cytokines IL-4 and IL-5 in asthmatic compared to normal bronchial mucosal MCs, again suggesting on-going activation.88-90 While most of the studies referred to above have been performed in atopic individuals, similar evidence supports a role for on-going MC activation in both non-atopic and occupational asthma.90-95

  *  !;    \; $"  #  It has been a long held view that the disordered airway physiology and airway wall #    #   ‹$  #  % { However, this view is no longer sustainable. Anti-IL-5 therapy depletes airway eosinophils by over 50% and while it reduces the rate of severe asthma exacerbations,117 it has no effect on day-to-day symptoms, BHR R or lung function. There are also many examples where # # % # {      % # is weak. This is most evident in patients with eosinophilic bronchitis (EB), a condition which accounts for about 15% of cases of cough referred to respiratory specialists. It is characterized by a corticosteroid responsive cough and the presence of a sputum eosinophilia $   #   !   {%  $  †`‚118 Interestingly the immunopathology of asthma and EB in the airway mucosa are virtually identical with   *^    #     ! $ @ $  IL-4 and IL-5 cytokine expression, epithelial integrity, sub-basement membrane collagen deposition and concentrations of histamine and PGD2.119-121 This suggests that many immunopathological features previously believed to contribute to asthma may not, in      # !   {%  $  †`‚119 MC numbers are not increased in the bronchial lamina propria of asthmatics compared  ## $‘ ˜* ’™ `%!  #    Œ <  #  98 (Fig. 2), the airway mucous glands,96 and the bronchial epithelium.88 For the ASM and glands this  ! @^    ! @ %# # $  % elements. This is likely to have profound effects on airway physiology (see below).

250

MAST CELL BIOLOGY

Figure 2.       #  % # $    %#  ^´! #

  ˜ %™ %  $      "’••

Mast Cell Microlocalization within Asthmatic Airway Smooth Muscle The striking difference between the pathology of asthma and EB lies in the ASM. Abnormal ASM function is critical in the pathophysiology of asthma and yet this structure had been largely neglected in previous immunopathological studies. In asthmatic subjects, there are numerous MCs within the ASM bundles, but virtually none in ASM from patients with EB or healthy subjects (Fig. 2).98 This is evident across disease phenotypes such as eosinophilic and non-eosinophilic subjects and across the spectrum of asthma severity.105,122 These ASM cells are activated and there is a correlation between the number of MCs within the ASM and the severity of BHR,98,105,123 suggesting that this is functionally relevant. The majority of MCs within the ASM are of the MCTC phenotype containing both tryptase and chymase and express IL-4 and IL-13 but not IL-5.98,101 In contrast we found that there were almost no T cells or eosinophils in the ASM in any  # $  $  $ #     # # $   $ MCs within the ASM is increased in fatal compared to nonfatal asthma,96 implicating MCs in the pathogenesis of asthma death. Interestingly, both MC numbers in the ASM and disease duration (e.g., prolonged MC presence within ASM) seem to contribute to BHR R in patients.105 *< #  #  !  #      MCs is a critical determinant of the asthmatic phenotype and have thus created a major paradigm shift in our understanding of asthma pathophysiology. However, this also has further implications. For example, not only might it clarify why many atopic patients do not have asthma, it would also explain why the presence of asthma is such a strong risk factor for death from anaphylaxis and allergen desensitization.124

MAST CELLS IN LUNG INFLAMMATION

251

Putative Mast Cell-Airway Smooth Muscle Interactions MCs produce many mediators that adversely affect ASM function (Tables 1-3). In many instances, cellular communication within the airways is likely to work across a distance of one or two microns rather than the thousands of microns involved in the depth of the lamina propria. The microlocalization of MCs within the ASM would therefore $$      % @       } mediator release and direct cell-cell contact. It is therefore entirely plausible that the presence of MCs within the ASM in asthma contributes to the development of ASM #  #  #  †`‚  !   {%  $ The ASM secretes many chemokines and growth factors which exhibit MC chemotactic activity, including CCL11, CXCL8 and CXCL12.11 However, the dominant ASM-dependent chemotactic pathway for HLMCs involves the CXCR3-CXCL10 axis. HLMCs express CXCR3 and cultured ASM from asthmatic subjects preferentially secretes the CXCR3 ligand CXCL10.5 The relevance of this to MC recruitment by the asthmatic ASM in vivo is demonstrated by the increased expression of CXCL10 by the ASM in bronchial biopsies from asthmatic compared to normal subjects and by the enrichment of CXCR3 MCs within the ASM bundles compared to the surrounding airway mucosa.5 The classical MC autacoid mediators histamine, PGD2 and LTC4 are all potent bronchoconstrictors. In mice, instillation of Th2 cell conditioned medium to the airways of naïve animals induced BHR R within 6 hours.125 This required expression of the IL-4 receptor alpha subunit and STAT6, suggesting a critical role for IL-4 and/or IL-13. We $    *  %# #   #98 but ASM MCs do express both IL-4 and IL-13.101 Exogenously administered tryptase induces bronchoconstriction and BHR R in dogs and sheep.126,127 In vitro, tryptase potentiates the contractile response of sensitized bronchi to histamine and induces proliferation of human ASM, but coculture of either resting or IgE/anti-IgE activated HLMCs with human ASM cells did not induce ASM proliferation.128-130 However, tryptase derived from HLMCs in coculture with human ASM induces the release of TGF` from ASM cells.131 This in turn increases the expression of _-smooth muscle actin in ASM cells, leading to increased histamine-dependent ASM contractility in collagen gels. These studies thus highlight several mechanisms through which MCs could exacerbate BHR R and promote bronchoconstriction. Contribution of MCs to local production of renin/angiotensin (Ang) in lungs has been recognized recently. Not only can MCs produce all components of a nontraditional renin-Ang system, including angiotensinogen, renin (which cleaves angiotensinogen into Ang I), chymase (which further converts Ang I into Ang II) and Ang II, but according to the data published by Veerappan et al, MCs are essential for local production of Ang II, which is a critical factor governing bronchoconstriction.132 In isolated bronchial rings in animal models, local generation of Ang II by MC renin elicits ASM contraction mediated by Ang II Type 1 receptors.132 Whether the components of this system are altered in asthmatic MCs and particularly those located within ASM remains to be determined. The majority of MCs (80%) in the ASM compartment belong to MCTC subtype.98 Chymase degrades human ASM ECM and inhibits T-cell adhesion to ASM, which might explain the paucity of T cells within this structure in asthma.133 Also of interest, a recent mouse model has suggested that mouse MC protease 4 (a homologue of human chymase) may actually offer protection against experimental antigen-induced airway {134 In this model, MCs were found in close proximity to ASM and absence

252

MAST CELL BIOLOGY

of MCP-4 resulted in ASM thickening in response to the OVA sensitization and challenge, clearly indicating a positive role MCP-4 in ASM remodeling. The communication between ASM cells and HLMCs is clearly bi-directional. In coculture HLMCs adhere to human ASM cells in part via cell adhesion molecule 1 (CADM1).135 HLMCs in coculture with ASM not only survive in the absence of exogenous survival factors, but proliferate rapidly.136 This is mediated through a co-operative interaction between CADM1, membrane-bound SCF expressed on ASM cells and soluble IL-6. Interestingly there is evidence for physical interaction in MCs between CADM1 and KIT, suggesting that CADM1 plays an important role in KIT function.136 Our hypothesis is that the initial adhesion mediated through CADM1 facilitates the interaction of MC KIT with ASM membrane-bound SCF. Another feature of this interaction is that HLMCs exhibit increased constitutive histamine release but can still respond to IgE/ anti-IgE activation.136 This indicates the presence of an ASM-dependent mechanism for the activation of HLMCs and could explain the presence of chronically activated MCs within the ASM bundles in asthma.  $  #     #   @  #$ #   $‹$ $  of asthma and likely to have a central role in the pathophysiology of ASM dysfunction. There is bi-directional cross-talk between these two cell types, with evidence to suggest the presence of a positive feedback loop between MC activation and disordered ASM #      #  % {    †`‚ ˜ Œ™ Mast Cell Microlocalization within Airway Epithelium and Submucosal Glands

@    #  # #$  #87,88,137 This is likely to be of importance in disease pathogenesis for two reasons. Firstly, this places MCs at the portal of entry of noxious stimuli such as aeroallergens, which would facilitate an effector role in the on-going immunological response (antigen presentation, Th2 cell differentiation, IgE synthesis). Furthermore, since tryptase degrades respiratory allergens and MC IgE on Fc¡RI is cross-linked by allergen which may be followed by tryptase release, there may   @^ ! < #     {138,139 Secondly, there are likely to be important consequences of MC activation on epithelial function. For example, MCs adhere avidly to bronchial epithelial cells and tryptase stimulates airway epithelial IL-8 release and ICAM-1 expression.140,141 However, coculture of MCs with the BEAS-2B bronchial epithelial cell line actually inhibits IgE-dependent HLMC degranulation.142 Whether this extends to chemokine and cytokine release is not known and awaits further work. One explanation is that normal airway epithelium keeps MCs “in check”, but once the epithelium is injured, this inhibitory activity is lost and MCs are then “free” to respond to the tissue insult. Severe mucus plugging is a well known feature of severe fatal asthma, but is also recognized as a feature of milder disease,143 and occurs as a result of mucus hypersecretion by hyperplastic submucosal glands and epithelial goblet cells. Like the ASM, mucous  #!      %#   #     {   We had noted previously that IL-6 MCs appeared abundant amongst airway mucosal glands.88 However, the work of Carroll and coworkers has provided strong evidence that

@ <     $  $ $  # *#    a detailed analysis of cartilaginous airways in post mortem lung specimens from patients with fatal asthma, patients with asthma who died from other causes (nonfatal asthma) and subjects without asthma who died of nonpulmonary causes.106 The number of MCs

Figure 3. Summary of important interactions between mast cells and structural airway cells in asthma.

MAST CELLS IN LUNG INFLAMMATION 253

254

MAST CELL BIOLOGY

     $ %     %# # $ gland stroma in nonfatal asthma and a marked increase in the number of degranulated MCs was present in both fatal asthma and nonfatal asthma compared to normal controls. Importantly, the density of both intact and degranulated MCs within the mucous glands correlated strongly with the degree of mucus obstruction in the airways, again implicating MCs in the pathogenesis of asthma death. Numerous MC mediators affect glandular growth and secretion including histamine, PGD2, LTC4, IL-6, TNF_ and chymase.53 Amphiregulin, a member of the epidermal growth factor family, is a further MC-derived molecule of particular interest with respect to the airway epithelium and mucosal glands. Amphiregulin expression is upregulated in human MCs following activation via IgE/anti-IgE,103,144 and this is not inhibited by dexamethasone. MCs resident in the asthmatic bronchial mucosa demonstrate increased amphiregulin expression; and in vitro, MC-derived amphiregulin increases mucin gene expression in the NCI-H292 epithelial cell line.103 Amphiregulin is present in increased concentrations in asthmatic sputum during asthma exacerbations; and it is proposed that this may aggravate airway wall remodeling.145 These observations suggest that MC-derived amphiregulin may play an important role in goblet cell hyperplasia and mucus hypersecretion in asthma and that this is resistant to the effects of corticosteroids. In addition, recombinant amphiregulin $ #     #$  %   $    $  $ # # %#  @   $  $#   $  Πsummarizes some potential interactions between MCs and structural airway cells in asthma.

#  (@    "  # The mechanisms of chronic MC activation in asthma are not fully understood. It is often assumed that allergen is the dominant factor resulting in cross-linking of Fc¡RI, but although allergen exposure is strongly linked to the development of asthma, once established, allergen avoidance usually has a minor effect on the state of established disease which appears to become “selff perpetuating”. This is most evident in cases of occupational asthma, where asthma and accompanying MC activation will persist on removal of the sensitizing agent if exposure is not prevented early in the course of the disease. MCs can be activated by a number of diverse stimuli including monomeric IgE alone, proteases, cytokines (e.g., SCF, TNF_, IFNa), complement, adenosine, TLR ligands, neuropeptides, hyperosmolality and cell contact (Fig. 1). It is likely that many  # #%    # " {  $  # #  % Monomeric IgE induces rodent MC mediator release and prolongs MC survival through the autocrine release of cytokines.146-149 In HLMCs IgE alone induces the release of histamine, LTC4 and IL-8 and prolongs MC survival after the withdrawal of SCF and IL-6 through autocrine secretion of IL-6.150,151 Importantly, while free IgE is present,  $  $ $ # #    # ! This is interesting because in humans there is a reproducible correlation between total serum IgE concentration, BHR R and asthma.152-155 Thus, heightened MC activation may arise in part from the increased binding of IgE to Fc¡RI. This is supported to some extent  #  ! # ^= #   < $ #  % {  MC activation as manifest by reduced IL-4 expression.156 However, anti-IgE therapy has a minimal effect on BHR, suggesting that monomeric IgE-dependent activation of MCs in asthmatic ASM does not account for this aspect of airway dysfunction.

MAST CELLS IN LUNG INFLAMMATION

255

SCF primes MCs for mediator release and at higher concentrations, directly induces degranulation.157 Concentrations of SCF are elevated in asthmatic airways and could therefore make an important contribution to ongoing MC activation.158,159 Interestingly, @  = #!      #    `2-adrenoceptor agonists, used as reliever medication for asthma, with respect to HLMC function. When applied to HLMCs acutely in vitro in the absence of SCF, they inhibit IgE/anti-IgE-dependent degranulation.69 However, after sustained exposure to the drug, rapid desensitization $  %#    160 Similarly, in vivo, acute administration of salbutamol and the long-acting `2-adrenoceptor agonist salmeterol to asthmatic subjects inhibit the airway response to allergen challenge. However, if these drugs are administered regularly for 1 week, not only is the protection against the EAR R lost, the magnitude of the EAR and the accompanying MC mediator release are enhanced.161,162 Furthermore, regular administration of short-acting `2-adrenoceptor agonists to asthmatic subjects has been associated with loss of asthma control.163,164 When HLMCs are incubated with SCF, their ability to respond to `2-adrenoceptor agonists is lost within 2 minutes.165 This is caused by the rapid uncoupling of the `2-adrenoceptor in HLMCs due to phosphorylation of Tyr350 on this receptor and subsequent internalization. Furthermore, in the presence of both SCF and IgE, salbutamol produced a dose-dependent increase in histamine release. *# # # " # !    #   !! #   @  # #    `2-adrenoceptor agonists and improve asthma control. Further evidence that this might occur in vivo comes from a study of asthma death, which demonstrates that the extent of MC degranulation correlates with blood salbutamol concentrations.166 The C5a receptor CD88 is expressed on the MCTC subset of HLMCs.10 Elevated @   #!     # $ $$  # $‘167 thus providing a further potential means of MC activation and of particular relevance to those MCs (MCTC) within the ASM bundles. As described above, HLMCs express the TLR-1-5, -7 and -9,47 stimulation of which results in MC activation. Perhaps of most relevance to asthma is MC activation via TLR3 with double-stranded viral RN R A ligand. Poly I:C, a synthetic activator of TLR3, $ #     ~_   "$   ‚ž  {$} ! $35 Since viruses are a common cause for asthma exacerbations, the MC antiviral response may be an important contributor to the deteriorating airway physiology. Nakayama and ^$#  ! #   *ƒ‚’  @     % { $

@^  W/Wv mice and TLR4
@  "   % {  $ # 169 In vitro studies revealed that LPS treatment augmented levels of IL-5 and -13, as well as mRN R A expression of IL-4, IL-5 and IL-13 and CCL24 (eotaxin-2) in wild-type bone marrow MCs, whereas no increase in IL-5 and eotaxin-2 was detected the case of TLR4
256

MAST CELL BIOLOGY

by increase in the levels of GATA1 transcription factor responsible for activation of the IL-4, IL-5 and IL-13 promoters. Furthermore, examination of the gene expression      #$  #   @ $ ##  $   arrays following activation with LPS via TLR4 compared to anti-IgE demonstrates # # $     $  ƒ\  ^=        expression.170 ƒ\^$ @   $  $   # $ a Th2 cytokine and chemokines that recruit Th2 cells and eosinophils.170 These data indicated that TLR4 in MCs may be involved in LPS-mediated enhancement of allergic  % { MC TLRs also have the potential to improve asthma. Pretreating mice with the TLR9 ligand, immunostimulatory sequences (ISS) of DNA containing a CpG motif,   # # $$    # @  # "   ƒ^’ and IL-9 in lung, as well as reduced the increased airway hyperreactivity to methacholine; all caused by chronic antigen challenge.48 In vitro experiments on MCs derived from the bone marrow of these mice showed that ISS did not affect histamine release, but induced IL-6 production.48 *#  $ # $  *ƒ‚^€  #!   effect in asthma model. Thymic stromal lymphopoetin is a cytokine released from airway epithelial cells in response to injury.171,172 Its overexpression in mice leads to asthmatic-type Th2-driven airway changes, while its inhibition attenuates the airway changes seen in mouse models of asthma.173 One paradigm for the effects of TSLP is that it activates CD11c dendritic cells which in turn leads to the differentiation of CD4 T cells into the Th2 phenotype through an interaction of OX40 with its ligand.174,175 However, it is well documented # *^^ #%   $  *ƒ\^ {  particular, the direct interaction of TSLP with MCs is highly relevant to the development   ^  % { *ƒ\  !   #$ #  ! with cytokines, peptidoglycan or trauma, induces the release of numerous cytokines and chemokines from human MCs, including IL-1.172 IL-13 production by MCs is inhibited when endogenous TSLP released by activated human epithelial cells is blocked.172 Furthermore, in a mouse model of rhinitis, MC activation was required for the increased TSLP production by airway epithelium.176 MC-derived TSLP also has the potential to  $  # !   % {104 TSLP is overexpressed in asthmatic airways,177,178 and so both MC-derived and epithelial-derived TSLP may be a key cytokine driving the MC in asthma. Immunoglobulin (Ig) free light chains are present in serum in normal subjects and #  $  $  {   $#  #$  In mice Ig free light chains can confer MC-dependent hypersensitivity through an unknown mechanism,179  ^  # #   @^ bronchoconstriction following antigen challenge.180 Concentrations of Ig free light chains are elevated in the sera of asthmatic compared to normal subjects suggesting they may be relevant to the pathophysiology of human asthma.180 Genetic factors may also lower the threshold for MC activation in asthma and thus predispose to the development of the disease and/or its severity. For example, an important negative regulator of MC activation is the phosphatase SHIP.181 `\^  MCs exhibit markedly enhanced secretory responses and with respect to human #    #   ‰# Š    !   of this molecule.182

MAST CELLS IN LUNG INFLAMMATION

257

   +   !; +`## Conventionally, MC activation in asthma has been interpreted as an IgE-dependent event, but recent studies indicate that common allergens, many of which are serine proteases, can induce MC histamine and cytokine secretion directly through an IgE-independent mechanism.183,184 This suggests that MCs may not only contribute to the chronic airway {     $  #!      #  of the allergic immune response, i.e., providing signals inducing B-cell IgE synthesis and Th2 lymphocyte differentiation.185,186

   #   # Several models have been developed which aim to induce the airway features of asthma. The most widely reported is the mouse model using intraperitoneal antigen sensitization followed by antigen challenge of the airways. This most closely resembles the model of acute allergen challenge in the airways although the route of sensitization is obviously different. The dependency on MCs in these models with regard to the !   % # !  {  #  #  % is highly dependent on the model studied and the mode of antigen sensitization. Thus sensitization without adjuvant generates a MC-dependent model while sensitization with adjuvant creates a MC-independent model.187 An alternative model uses airway sensitization without adjuvant from the outset and, to some extent, this is more physiological. In this setting, MCs are again an essential  ‹$   # !   % # ! { and remodeling (collagen deposition, goblet cell hyperplasia.188-190 Furthermore, mouse models allowed to estimate contribution of infections and TLR R activation, as well as the nervous system42,168,169,191,192 to MC function in the development of asthma. However, there are inevitably a number of problems in relating these models to the human disease. For example, mouse airways contain very few MCs at baseline, so the changes seen following antigen challenge rely heavily on the recruitment of MC progenitors rather than the activity of resident cells. Therefore, it is perhaps not surprising that short term models using intraperitoneal sensitization do not reveal a role for MCs in the outcomes commonly measured. In addition, mice have relatively little smooth muscle in their airways and so there is no model described to date in mice which has recapitulated the       @–  $  #   <  # !  # in humans. So while mouse models are useful for generating hypotheses regarding the #  # #       Mast Cells in Chronic Obstructive Pulmonary Disease (COPD) @ˆ\   #  $  # % %  #  }   {%  #    "  %##  $$   ! *#   %#    {    # $  "$       < {  %      %#  % #< %– #   %      $  !     # %#  there is overwhelming evidence for MC involvement, their role in COPD is less obvious (Table 4). In COPD patients, the MC population in the lung undergoes changes in density and distribution related to a severity of the disease. In contrast to asthma, the total MC

258

MAST CELL BIOLOGY

density in lung, as well as MCT in submucosa, progressively decreases compared with healthy lung, but MC activation and degranulation are increased as in asthma, coinciding with enhanced histamine release in advanced COPD.107,193 Similar to asthma, an increased MCTC density has been reported in alveolar parenchyma and in ASM.107,109 In lungs from patients with very severe COPD, there is a several-fold increase in the MCTC/MC ratio cells in small airways, small airway epithelium, pulmonary vessels and alveolar parenchyma, with MCTC comprising ¾95% of the total MCs in larger airways.107 Of particular importance is the increased CD88 expression in both MCTC and MCT cells,107 which coincides with increased expression of the CD88 ligand c5a in COPD patients167 and which is likely to cause MC activation. The changes in MCTC population also coincide with increased expression of TGF` and renin, but their functional importance in COPD has not been elucidated. Interestingly, increased MCT and MCTC numbers positively correlate with the lung function in COPD.109 High levels of MC activation in COPD are also likely to be caused by elevated bronchial mucosal expression of TSLP and chemokines similar to that found in severe asthma.177 Mast Cells in Interstitial Lung Disease   $    #  }  #    $    occurring as a result of injury and aberrant repair. The most common form of pulmonary

  $   #   # $    ˜\™ %#  #    $$   $ ˜ <%  !$      !™ ˜ !%    €’™ &# #  !    { in the onset of IPF remain controversial, there is good evidence of ongoing damage to the alveolar epithelium, basement membrane and capillary endothelium, leading to the !   # %#      @  # {  cells. The chronicity of the insult leads to dysregulated repair with the generation of

   *# !    !  !         < including TGF`1, PDGF and bFGF.194 *# <   ! # !     #    %## #    # %    # muscle, in that it expresses _-smooth muscle actin (_SMA) and exhibits contractile ! $    # #    "!    "195 The     #    ##  !    #    \    $    #      @ $  $ $   %# #    $ %# @ $     %# #   

  ˜* ’™110 In lungs of patients with idiopathic bronchiolitis obliterans organizing pneumonia, MC density and activation are also increased.112 In addition increased MC density, with MCTC    $$         $ in idiopathic interstitial pneumonia.113  #      $ # ‘  

@     # !    196 A marked increase in MC density  @ #  #     # #<  $ !   % as in MCs also were found within the alveolar epithelial layer and alveolar lumina of  %# $    $   !  !       control specimens, in which MCs were most frequently seen in subpleural and perivascular connective tissue.111 These MCs often show reduced numbers of granules and disorganized granule content, suggesting that there is partial ongoing degranulation. In addition, about 10-fold-elevated histamine concentrations have been described in BAL from patients with IPF.197    $   \  # $  ^"    $

MAST CELLS IN LUNG INFLAMMATION

259

 $ %## #!     @114-116 These bFGF-containing MCs are $ $       # $   ^< 116 Furthermore, the distribution of bFGF-containing MCs matches that of ECM deposition    %# # "   –  #   !  $ ! # @  $  #     Alveolar hypoxia reportedly induces activation of MCs, leading to local generation     ‹$   {198 MC stabilization with cromolyn prevented the systemic response, as did ACE inhibitors and AngII receptor blockers, through interactions between leukocytes and endothelial cells. It is conceivable that  #  ! @  $     < @ ! %#  {  @ˆ\        %# @ through cell-cell adhesion thereby inducing the release of various MC-derived mediators.  " $$     <   #}  @  $  this is followed by secretion of collagenase and `-hexosaminidase.199 Histamine, TNF_, bFGF, TGF`  ƒ^’         #$200-204 In coculture, human

@ #  !  #$   # $# $   205 MCs augment     #$ <   ! @^ ! ƒ^’   ^ contact.206 However, IL-4 could not be detected in coculture, suggesting that IL-4 was    @  % $      ^  %#   We proposed that MCs may present IL-4 on their surface in order to confer local cytokine  207 ƒ^’   #   #$     $ #$

     =@  208,209 Among HLMC-derived mediators, bFGF is likely to have a major effect on lung

  `  }    $$     !  !    it from degradation.210 Furthermore, heparin and/or heparan sulfate are required for binding of bFGF to its receptors211 and also release it from basement membranes where it is stored.212 `   #$    !       through the regulation of bFGF activity. In addition, MC tryptase induces proliferation  $  #   %   ! !  \‚^_213 In animal models, increased numbers of MCs and elevated lung histamine content have  $  $    $     214-216  # $    ^$ $  { @ #     $ @^   !    !  $   #!  reduced BAL neutrophilia and protein content.217 MCs are also implicated in pulmonary capillary hemangiomatosis, causing pulmonary arterial hypertension with no effective medical therapy. Microarray analysis of lesions    %# #    #   ! "   @^    surfactant protein C and tryptase.218 @    @” $   an abundance of MCs with high PDGFR-` expression in this disease. Lung Cancer The role of MCs in cancer is discussed in more detail in Chapter 14. Lung cancer is the leading cause of death from malignancy worldwide with nonsmall cell lung cancer (NSCLC) accounting for the majority of these cases. There is extensive evidence indicating that the immune system plays a key role in the regulation of cancer development and progression,219 and cells of the innate and adaptive immune responses have been implicated in both the progression and curtailment of tumor growth.220 The role of MCs in tumor

260

MAST CELL BIOLOGY

development and progression is controversial due to the nature of the mediators they generate.221 Several lines of evidence have suggested that MCs promote tumor progression and metastasis,222-224 potentially through their ability to promote angiogenesis via the release of autacoid mediators and pro-angiogenic chemokines and growth factors.225,226 For example, the granular products of MCs, histamine and cannabinoids released during degranulation, have been demonstrated in coculture to enhance the migration of cervical cancer cells.227 Recently a peptide adrenomedullin implicated in cross-talk between MCs and cancer cells was localized in MCs within lung cancer.228 Increased MC numbers have been documented to correlate with poor outcome in patients with NSCLC, especially lung adenocarcinoma.222-224 and microvessel density,222-224,229-233 although exceptions have been noted.234,235 Nevertheless, other studies found positive correlations between MC intratumoral density and good prognosis,230,236,237 particular at        # @TC type.233,234 MC counts also negatively correlate with the presence of metastatic lung cancer in lymphatic nodes.238 There are however reports of lack of correlation between lung cancer survival and MCs.232,235,239 Regardless of their

    %# # $  #   #    #   MC within NSCLC tumors. We therefore carefully examined the density of tryptase MC in the tumor stroma compared to the clusters of tumor islets in 175 patients with surgically resected NSCLC. These studies revealed that MC numbers were similar in the tumor stroma of patients with surgically resected NSCLC irrespective of survival status, but there was a marked survival advantage when MC were present in NSCLC tumor islets.237 Therefore, as in other locations and diseases, the microanatomical location of MC is likely to be key with respect to their biological activity within the tumor microenvironment.

CONCLUSION There is overwhelming evidence, based largely on studies from humans, that MCs play a central role in asthma pathogenesis and are undoubtedly involved in many diverse pulmonary diseases. The goal of current research is to understand the mechanisms driving their chronic activation and cellular cross-talk, with a view to developing novel therapies which are effective when administered to patients long-term in vivo.

REFERENCES 1. Weller CL, Collington SJ, Hartnell A et al. Chemotactic action of prostaglandin E2 on mouse mast cells acting via the PGE2 receptor 3. Proc Natl Acad Sci USA 2007; 104:11712-7. 2. Jones TG, Hallgren J, Humbles A et al. Antigen-induced increases in pulmonary mast cell progenitor numbers depend on IL-9 and CD1d-restricted NKT cells. J Immunol 2009; 183:5251-60. 3. Roviezzo F, D’Agostino B, Brancaleone V et al. Systemic Administration of Sphingosine-1-phosphate Increases Bronchial Hyper-responsiveness in the Mouse. Am J Respir Cell Mol Biol 2009; [Epub ahead of print]. 4. Weller CL, Collington SJ, Brown JK K et al. Leukotriene B4, an activation product of mast cells, is a chemoattractant for their progenitors. J Exp Med 2005; 201:1961-71. 5. Brightling CE, Ammit AJ, Kaur D et al. The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle. Am J Respir Crit Care Med 2005; 171:1103-8. 6. Bradding P. Human lung mast cell heterogeneity. Thorax 2009; 64:278-80. 7. Weidner N, Austen KF. Heterogeneity of mast cells at multiple body sites. Fluorescent determination of !   ${$     #     "  Pathol Res Pract 1993; 189:156-62.

MAST CELLS IN LUNG INFLAMMATION

261

8. Irani AM, Goldstein SM, Wintroub BU et al. Human mast cell carboxypeptidase. Selective localization to MCTC cells. J Immunol 1991; 147:247-53. 9. Bradding P, Okayama Y, Howarth PH et al. Heterogeneity of human mast cells based on cytokine content. J Immunol 1995; 155:297-307. 10. Oskeritzian CA, Zhao W, Min HK K et al. Surface CD88 functionally distinguishes the MCTC from the MCT type of human lung mast cell. J Allergy Clin Immunol 2005; 115:1162-8. 11. Brightling CE, Kaur D, Berger P et al. Differential expression of CCR3 and CXCR3 by human lung and bone marrow-derived mast cells: implications for tissue mast cell migration. J Leukoc Biol 2005; 77:759-66. _  ` $  ˆ<$$      "      #$   $   silico study. Allergol Int 2006; 55:173-9. 13. Balzar S, Chu HW, Strand M et al. Relationship of small airway chymase-positive mast cells and lung function in severe asthma. Am J Respir Crit Care Med 2005; 171:431-9. 14. Schulman ES, Kagey-Sobotka A, MacGlashan DW, Jr. et al. Heterogeneity of human mast cells. J Immunol 1983; 131:1936-41. 15. Schulman ES, Pollack RB, Post TJ et al. Histochemical heterogeneity of dispersed human lung mast cells. J Immunol 1990; 144:4195-201. “   @    †‘  ƒ   ~! ^    $$  # #$ $ Thorax 2009; 64:297-305. ” ## ~       % >ƒ       # ^<    # @*@ type of human mast cell. J Immunol 1990; 145:2652-61. 18. Romagnani P, De Paulis A, Beltrame C et al. Tryptase-chymase double-positive human mast cells express the eotaxin receptor CCR3 and are attracted by CCR3-binding chemokines. Am J Pathol 1999; 155:1195-204. 19. Irani AM, Bradford TR, Kepley CL et al. Detection of MCT and MCTC types of human mast cells by immunohistochemistry using new monoclonal anti-tryptase and anti-chymase antibodies. J Histochem Cytochem 1989; 37:1509-15. 20. Hsieh FH, Sharma P, Gibbons A et al. Human airway epithelial cell determinants of survival and functional phenotype for primary human mast cells. Proc Natl Acad Sci USA 2005; 102:14380-5. 21. Mierke CT, Ballmaier M, Werner U et al. Human endothelial cells regulate survival and proliferation of human mast cells. J Exp Med 2000; 192:801-11. 22. Malaviya R, Ross EA, MacGregor JI et al. Mast cell phagocytosis of FimH-expressing enterobacteria. J Immunol 1994; 152:1907-14. _Œ ! ‚ < * ‚ =     $  $ # {$"        of infection through TNF-alpha. Nature 1996; 381:77-80. 24. Sutherland RE, Olsen JS, McKinstry A et al. Mast cell IL-6 improves survival from Klebsiella pneumonia and sepsis by enhancing neutrophil killing. J Immunol 2008; 181:5598-605. 25. Xu X, Zhang D, Lyubynska N et al. Mast cells protect mice from Mycoplasma pneumonia. Am J Respir Crit Care Med 2006; 173:219-25. 26. Hoek KL, Cassell GH, Duffy LB et al. Mycoplasma pneumoniae-induced activation and cytokine production in rodent mast cells. J Allergy Clin Immunol 2002; 109:470-6. 27. Luo D, Dai Y, Duffy LB et al. Inhibition of message for FcepsilonRI alpha chain blocks mast cell IL-4 production induced by coculture with Mycoplasma pneumoniae. Microb Pathog 2008; 44:286-92. 28. Laichalk LL, K Kunkel SL, Strieter RM et al. Tumor necrosis factor mediates lung antibacterial host defense in murine Klebsiella pneumonia. Infect Immun 1996; 64:5211-8. _€  ‚  ! >>  # >>   ‚  {       $  pneumococcal infection. Infect Immun 2002; 70:1547-57. 30. Ketavarapu JM, Rodriguez AR, Yu JJ et al. Mast cells inhibit intramacrophage Francisella tularensis replication via contact and secreted products including IL-4. Proc Natl Acad Sci USA 2008; 105:9313-8. 31. Thakurdas SM, Melicoff E, Sansores-Garcia L et al. The mast cell-restricted tryptase mMCP-6 has a critical immunoprotective role in bacterial infections. J Biol Chem 2007; 282:20809-15. Œ_ `$ @   * ˆ§†  \>   =!$  # $     #$   tryptase beta I and demonstration of its importance in bacterial infections of the lung. J Biol Chem 2001; 276:26276-84. ŒŒ #  ~ ! \ @ # >      $  $$ {   #  ! # tryptase/heparin complexes. J Immunol 2009; 182:647-56. 34. Alho OP, Karttunen TJ, Karttunen R et al. Lymphocyte and mast cell counts are increased in the nasal mucosa in symptomatic natural colds. Clin Exp Immunol 2003; 131:138-42. 35. K Kulka M, Alexopoulou L, Flavell RA et al. Activation of mast cells by double-stranded RN R A: evidence for activation through Toll-like receptor 3. J Allergy Clin Immunol 2004; 114:174-82. 36. Shirato K, Taguchi F. Mast cell degranulation is induced by A549 airway epithelial cell infected with respiratory syncytial virus. Virology 2009; 386:88-93.

262

MAST CELL BIOLOGY

Œ” <#  \ < >& *$ @   *#   ! $^  $$ =   % hyperresponsiveness. Am J Respir Crit Care Med 2004; 170:952-9. 38. Heib V, Becker M, Taube C et al. Advances in the understanding of mast cell function. Br J Haematol 2008; 142:683-94. Œ€ &^†  `$ @ ‚ $}

  ƒ$<   $  {  $  $ rats infected with respiratory syncytial virus. Am J Physiol Lung Cell Mol Physiol 2002; 282:L1143-50. 40. Becker Y. Respiratory syncytial virus (RSV) evades the human adaptive immune system by skewing the Th1/Th2 cytokine balance toward increased levels of Th2 cytokines and IgE, markers of allergy—a review. Virus Genes 2006; 33:235-52. ’ $%   `# @ &#    %# {$}  ! $   {$ ^$ cutaneous anaphylaxis in mice. J Invest Dermatol 2002; 118:645-51. ’_ <#  ƒ š  ˆ## `   ž $^  = #  % !   with respiratory syncytial virus in newborn mice. J Allergy Clin Immunol 2009; 123:138-45 e5. ’Œ ! > < ž $ $ ž\ ^     $  $ infection. Ann Allergy Asthma Immunol 1996; 77:67-73. 44. Kjaergard K LL, Larsen FO, Norn S et al. Basophil-bound IgE and serum IgE directed against Haemophilus {$}   $ $   %# #   # $  $ "  Apmis 1996; 104:61-7. 45. Emre U, Sokolovskaya N, Roblin PM et al. Detection of anti-Chlamydia pneumoniae IgE in children with reactive airway disease. J Infect Dis 1995; 172:265-7. ’“  ~ * ` <}%        "     %    progenitor-derived and adult progenitor-derived human mast cells. Immunol Lett 2005; 98:265-71. ’” $<    *ƒ‚Œ ! # #$   #     !  Mol Immunol 2006; 43:1579-86. 48. Ikeda RK, Miller M, Nayar J et al. Accumulation of peribronchial mast cells in a mouse model of ovalbumin   $ #   % { $  $$  ~ ‹$ J Immunol 2003; 171:4860-7. 49. Yoshioka M, Fukuishi N, Iriguchi S et al. Lipoteichoic acid downregulates FcepsilonRI expression on human mast cells through Toll-like receptor 2. J Allergy Clin Immunol 2007; 120:452-61. 50. Zaidi AK, Thangam ER, Ali H. Distinct roles of Ca2 mobilization and G protein usage on regulation of Toll-like receptor function in human and murine mast cells. Immunology 2006; 119:412-20. 51. Carlos D, Frantz FG, Souza-Junior DA et al. TLR2-dependent mast cell activation contributes to the control of Mycobacterium tuberculosis infection. Microbes Infect 2009; 11:770-8. 52. Bradding P, Walls AF, Holgate ST. The role of the mast cell in the pathophysiology of asthma. J Allergy Clin Immunol 2006; 117:1277-84. 53. Bradding P. Mast cells in asthma. In: Busse WW, Holgate ST, eds. Asthma and rhinitis. Boston: Blackwell   \$  _•••Œ€^Œ— 54. Newson R, Strachan D, Archibald E et al. Effect of thunderstorms and airborne grass pollen on the incidence of acute asthma in England, 1990-94. Thorax 1997; 52:680-5. 55. Newson R, Strachan D, Archibald E et al. Acute asthma epidemics, weather and pollen in England, 1987-1994. Eur Respir J 1998; 11:694-701. “ # ‚\  # & >  \  \   @#  }  {      $   #$ $    ‚! ‚  €—“– ŒŒ“’^” ” @ *† &  ‚#  `†   =!  #! ! {$ # !  allergic asthmatics are associated with methacholine bronchial hyperresponsiveness. J Clin Invest 1987; 79:1197-203. 58. Wenzel SE, Fowler AA, 3rd, Schwartz LB. Activation of pulmonary mast cells by bronchoalveolar allergen challenge. In vivo release of histamine and tryptase in atopic subjects with and without asthma. Am Rev Respir Dis 1988; 137:1002-8. 59. Murray JJ, Tonnel AB, Brash AR R et al. Release of prostaglandin D2 into human airways during acute antigen challenge. N Engl J Med 1986; 315:800-4. “• &} = ƒ  ƒ >#    =! !  $<  @’   #! ! {$ from atopic asthmatics after endobronchial allergen challenge. Am Rev Respir Dis 1990; 142:112-9. “ &} = & >š ƒ  ƒ † #! ! {$  !  $    challenge in atopic subjects with asthma: relationship to the development of late asthmatic responses. J Allergy Clin Immunol 1991; 87:540-8. “_ ƒ$ @ `$  &@ \ $       {    %  challenge of the peripheral airways in allergic asthmatics. Cellular, mediator and permeability changes. Am Rev Respir Dis 1991; 144:51-8.

MAST CELLS IN LUNG INFLAMMATION

263

63. Sedgwick JB, Calhoun WJ, Gleich GJ et al. Immediate and late airway response of allergic rhinitis patients to segmental antigen challenge. Characterization of eosinophil and mast cell mediators. Am Rev Respir Dis 1991; 144:1274-81. 64. Rafferty P, Beasley R, Holgate ST. The contribution of histamine to immediate bronchoconstriction provoked by inhaled allergen and adenosine 5v monophosphate in atopic asthma. Am Rev Respir Dis 1987; 136:369-73. “ @$ } ~ ‚  \ ` * =   ^" #  {$     ` histamine receptor antagonist, terfenadine, alone and in combination on allergen induced immediate bronchoconstriction in man. Thorax 1987; 42:946-52. 66. Taylor IK, O’Shaughnessy KM, Fuller RW et al. Effect of cysteinyl-leukotriene receptor antagonist ICI 204.219 on allergen-induced bronchoconstriction and airway hyperreactivity in atopic subjects. Lancet 1991; 337:690-4. 67. Findlay SR, Barden JM, Easley CB et al. Effect of the oral leukotriene antagonist, ICI 204,219, on antigen-induced bronchoconstriction in subjects with asthma. J Allergy Clin Immunol 1992; 89:1040-5. 68. Beasley R, Varley J, Robinson C et al. Cholinergic-mediated bronchoconstriction induced by prostaglandin D2, its initial metabolite 9 alpha,11 beta-PGF2 and PGF2 alpha in asthma. Am Rev Respir Dis 1987; 136:1140-4. 69. Church MK, Hiroi J. Inhibition of IgE-dependent histamine release from human dispersed lung mast cells by anti-allergic drugs and salbutamol. Br J Pharmacol 1987; 90:421-9. 70. Pepys J, Hargreave FE, Chan M et al. Inhibitory effects of disodium cromoglycate on allergen-inhalation tests. Lancet 1968; 2:134-7. ” `% # \` $ # ‚ ƒ *`   {$  $    $    $   on the airway and circulating mediator responses to allergen bronchial provocation in asthma. Am Rev Respir Dis 1985; 132:986-92. 72. Fahy JV, Fleming HE, Wong HH et al. The effect of an anti-IgE monoclonal antibody on the early- and late-phase responses to allergen inhalation in asthmatic subjects. Am J Respir Crit Care Med 1997; 155:1828-34. 73. Boulet LP, Chapman KR, Cote J et al. Inhibitory effects of an anti-IgE antibody E25 on allergen-induced early asthmatic response. Am J Respir Crit Care Med 1997; 155:1835-40. 74. De Monchy JG, Kauffman HF, Venge P et al. Bronchoalveolar eosinophilia during allergen-induced late asthmatic reactions. Am Rev Respir Dis 1985; 131:373-6. 75. Metzger WJ, Zavala D, Richerson HB et al. Local allergen challenge and bronchoalveolar lavage of   # $    #     % {  ‚! ‚  1987; 135:433-40. 76. Bentley AM, Meng Q, Robinson DS et al. Increases in activated T-lymphocytes, eosinophils and cytokine mRN R A expression for interleukin-5 and granulocyte/macrophage colony-stimulating factor in bronchial biopsies after allergen inhalation challenge in atopic asthmatics. Am J Respir Cell Mol Biol 1993; 8:35-42. ””    }$ @ $    † #  !  $<     upregulation of leukocyte-endothelial cell adhesion molecules 6 hours after local allergen challenge of sensitized asthmatic airways. J Clin Invest 1994; 93:1411-21. 78. Tonnel AB, Joseph M, Gosset P et al. Stimulation of alveolar macrophages in asthmatic patients after local provocation test. Lancet 1983; 1:1406-8. 79. Diaz P, Gonzalez MC, Galleguillos FR R et al. Leukocytes and mediators in bronchoalveolar lavage during allergen-induced late-phase asthmatic reactions. Am Rev Respir Dis 1989; 139:1383-9. —•   >  `  ! = # >   † #!      #  # subjects. Am Rev Respir Dis 1987; 136:379-83. 81. Flint KC, Leung KB, Hudspith BN et al. Bronchoalveolar mast cells in extrinsic asthma: a mechanism for #       #  †  > ˜@ ‚ =™ €—– _€€_Œ^“ 82. Broide DH, Gleich GJ, Cuomo AJ et al. Evidence of ongoing mast cell and eosinophil degranulation in symptomatic asthma airway. J Allergy Clin Immunol 1991; 88:637-48. 83. Casolaro V, Galeone D, Giacummo A et al. Human basophil/mast cell releasability. V. Functional comparisons of cells obtained from peripheral blood, lung parenchyma and bronchoalveolar lavage in asthmatics. Am Rev Respir Dis 1989; 139:1375-82. 84. Wardlaw AJ, Dunnette S, Gleich GJ et al. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma. Relationship to bronchial hyperreactivity. Am Rev Respir Dis 1988; 137:62-9. 85. Beasley R, Roche WR, Roberts JA et al. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 1989; 139:806-17. 86. Djukanovic R, Lai CK, Wilson JW et al. Bronchial mucosal manifestations of atopy: a comparison of  <   { %  #  #  ##   =$ ‚ J 1992; 5:538-44.

264

MAST CELL BIOLOGY

—” ƒ ƒ ƒ  `# *  % $ { !   %# %  asthma. Am Rev Respir Dis 1993; 147:697-704. 88. Bradding P, Roberts JA, Britten KM et al. Interleukin-4, -5 and -6 and tumor necrosis factor-alpha in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines. Am J Respir Cell Mol Biol 1994; 10:471-80. 89. Ying S, Durham SR, Corrigan CJ et al. Phenotype of cells expressing mRN R A for TH2-type (interleukin 4 and interleukin 5) and TH1-type (interleukin 2 and interferon gamma) cytokines in bronchoalveolar lavage and bronchial biopsies from atopic asthmatic and normal control subjects. Am J Respir Cell Mol Biol 1995; 12:477-87. 90. Ying S, Humbert M, Barkans J et al. Expression of IL-4 and IL-5 mRN R A and protein product by CD4

and CD8 T-cells, eosinophils and mast cells in bronchial biopsies obtained from atopic and nonatopic (intrinsic) asthmatics. J Immunol 1997; 158:3539-44. €        \    % $ {  $ # $ by toluene diisocyanate. Am Rev Respir Dis 1992; 145:160-8. €_ `$    > * ^†  ƒ   `#^  =  ˜‚™^    bronchial biopsies from atopic and nonatopic asthma. Am J Respir Crit Care Med 1996; 153:1931-7. 93. Di Stefano A, Saetta M, Maestrelli P et al. Mast cells in the airway mucosa and rapid development of occupational asthma induced by toluene diisocyanate. Am Rev Respir Dis 1993; 147:1005-9. €’ @#^š$  @# ` *    `  $<     #! {$ $  plicatic acid-induced bronchoconstriction. J Allergy Clin Immunol 1989; 84:762-8. 95. Frew A, Chan H, Dryden P et al. Immunologic studies of the mechanisms of occupational asthma caused by western red cedar. J Allergy Clin Immunol 1993; 92:466-78. 96. Carroll NG, Mutavdzic S, James AL. Distribution and degranulation of airway mast cells in normal and asthmatic subjects. Eur Respir J 2002; 19:879-85. 97. Chen FH, Samson KT, Miura K et al. Airway remodeling: a comparison between fatal and nonfatal asthma. J Asthma 2004; 41:631-8. €— † # @= †  \     ^      % # $  # N Engl J Med 2002; 346:1699-705. 99. Zanini A, Chetta A, Saetta M et al. Chymase-positive mast cells play a role in the vascular component of airway remodeling in asthma. J Allergy Clin Immunol 2007; 120:329-33. •• # @ $ * #     \$      {   # @ =" Allergy 2009; 39:1499-507. 101. Brightling CE, Symon FA, Holgate ST et al. Interleukin-4 and -13 expression is colocalized to mast cells within the airway smooth muscle in asthma. Clin Exp Allergy 2003; 33:1711-6. 102. Amin K, Janson C, Harvima I et al. CC chemokine receptors CCR1 and CCR4 are expressed on airway mast cells in allergic asthma. J Allergy Clin Immunol 2005; 116:1383-6. 103. Okumura S, Sagara H, Fukuda T et al. FcepsilonRI-mediated amphiregulin production by human mast cells increases mucin gene expression in epithelial cells. J Allergy Clin Immunol 2005; 115:272-9. 104. Okayama Y, Okumura S, Sagara H et al. FcepsilonRI-mediated thymic stromal lymphopoietin production by interleukin-4-primed human mast cells. Eur Respir J 2009; 34:425-35. 105. Siddiqui S, Mistry V, Doe C et al. Airway hyperresponsiveness is dissociated from airway wall structural remodeling. J Allergy Clin Immunol 2008; 122:335-41, 41 e1-3. 106. Carroll NG, Mutavdzic S, James AL. Increased mast cells and neutrophils in submucosal mucous glands and mucus plugging in patients with asthma. Thorax 2002; 57:677-82. 107. Andersson CK, Mori M, Bjermer L et al. Alterations in Lung Mast Cell Populations in Patients with COPD. Am J Respir Crit Care Med 2009. 108. Saha S, Mistry V, Siva R et al. Induced sputum and bronchial mucosal expression of interleukin-13 is not increased in chronic obstructive pulmonary disease. Allergy 2008; 63:1239-43. 109. Gosman MM, Postma DS, Vonk JM et al. Association of mast cells with lung function in chronic obstructive pulmonary disease. Respir Res 2008; 9:64. • \  †             $    @# €€Œ– •Œ€—€^€“  % ˆ   ž> $ >   '  $$   $      %#   lung disorders. Lab Invest 1979; 40:717-34. 112. Pesci A, Majori M, Piccoli ML et al. Mast cells in bronchiolitis obliterans organizing pneumonia. Mast cell hyperplasia and evidence for extracellular release of tryptase. Chest 1996; 110:383-91. 113. Hirata K, Sugama Y, Ikura Y et al. Enhanced mast cell chymase expression in human idiopathic interstitial pneumonia. Int J Mol Med 2007; 19:565-70. ’ Ÿ$   ƒ >  \%  ‚        ‘ $       %#   #  {  $$ #  > \# €€– ’”“’^”Œ  $ š  *= >  *<    `$       %#   $ 

      > \# €€“– ’€_•Œ”^’

MAST CELLS IN LUNG INFLAMMATION

265

“ $ š  *= >  † < =   †    %#       # $ 

   #  > ‚ @  @   _••_– ““”“^”Œ 117. Haldar P, Brightling CE, Hargadon B et al. Mepolizumab and exacerbations of refractory eosinophilic asthma. N Engl J Med 2009; 360:973-84. 118. Brightling CE, Ward R, Goh KL et al. Eosinophilic bronchitis is an important cause of chronic cough. Am J Respir Crit Care Med 1999; 160:406-10. € † # @= &  ‚ &   $ $$ {      eosinophilic bronchitis and asthma. Am J Respir Crit Care Med 2000; 162:878-82. _• † # @=   †     *`_ < "    #! ! {$ T-lymphocytes and bronchial submucosa is a feature of asthma and eosinophilic bronchitis. J Allergy Clin Immunol 2002; 110:899-905. 121. Brightling CE, Symon FA, Birring SS et al. Comparison of airway immunopathology of eosinophilic bronchitis and asthma. Thorax 2003; 58:528-32. 122. Berry M, Morgan A, Shaw DE et al. Pathological features and inhaled corticosteroid response of eosinophilic and non-eosinophilic asthma. Thorax 2007; 62:1043-9. _Œ †$  ` †  \ ž ‘$" >   {   # # $    # Thorax 2007; 62:8-15. 124. Bock SA, Munoz-Furlong A, Sampson HA. Fatalities due to anaphylactic reactions to foods. J Allergy Clin Immunol 2001; 107:191-3. 125. Venkayya R, Lam M, Willkom M et al. The Th2 lymphocyte products IL-4 and IL-13 rapidly induce airway hyperresponsiveness through direct effects on resident airway cells. Am J Respir Cell Mol Biol 2002; 26:202-8. 126. Sekizawa K, Caughey GH, Lazarus SC et al. Mast cell tryptase causes airway smooth muscle hyperresponsiveness in dogs. J Clin Invest 1989; 83:175-9. 127. Molinari JF, Scuri M, Moore WR R et al. Inhaled tryptase causes bronchoconstriction in sheep via histamine release. Am J Respir Crit Care Med 1996; 154:649-53. 128. Berger P, Perng DW, Thabrew H et al. Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells. J Appl Physiol 2001; 91:1372-9. 129. Brown JK, Jones CA, Rooney LA et al. Tryptase’s potent mitogenic effects in human airway smooth muscle cells are via nonproteolytic actions. Am J Physiol Lung Cell Mol Physiol 2002; 282:L197-206. 130. Kaur D, Hollins F, Saunders R et al. Airway smooth muscle proliferation and survival is not modulated by mast cells. Clin Exp Allergy 2009. 131. Woodman L, Siddiqui S, Cruse G et al. Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-beta 1. J Immunol 2008; 181:5001-7. 132. Veerappan A, Reid AC, Estephan R et al. Mast cell renin and a local renin-angiotensin system in the airway: role in bronchoconstriction. Proc Natl Acad Sci USA 2008; 105:1315-20. ŒŒ ƒ} ƒ \<  $# '     #   ^ "    # mitogen-induced proliferation of human airway smooth muscle cells. J Immunol 2002; 169:1014-20. 134. Waern I, Jonasson S, Hjoberg H J et al. Mouse mast cell protease 4 is the major chymase in murine airways  #   !      % { > $ _••€– —Œ“Œ“€^”“ 135. Yang W, Kaur D, Okayama Y et al. Human lung mast cells adhere to human airway smooth muscle, in part, via tumor suppressor in lung cancer-1. J Immunol 2006; 176:1238-43. 136. Hollins F, Kaur D, Yang W et al. Human airway smooth muscle promotes human lung mast cell survival, proliferation and constitutive activation: cooperative roles for CADM1, stem cell factor and IL-6. J Immunol 2008; 181:2772-80. 137. Pesci A, Foresi A, Bertorelli G et al. Histochemical characteristics and degranulation of mast cells in epithelium and lamina propria of bronchial biopsies from asthmatic and normal subjects. Am Rev Respir Dis 1993; 147:684-9. 138. Rauter I, K Krauth MT, Flicker S et al. Allergen cleavage by effector cell-derived proteases regulates allergic { =† > _••“– _•€“”^€ Œ€ ‚$   $# * & #     ^ !       { through cleavage of IgE. J Allergy Clin Immunol 2008; 121:197-202. 140. Sanmugalingam D, Wardlaw AJ, Bradding P. Adhesion of human lung mast cells to bronchial epithelium: evidence for a novel carbohydrate-mediated mechanism. J Leukoc Biol 2000; 68:38-46. 141. Cairns JA, Walls AF. Mast cell tryptase is a mitogen for epithelial cells. Stimulation of IL-8 production and intercellular adhesion molecule-1 expression. J Immunol 1996; 156:275-83. 142. Yang W, Wardlaw AJ, Bradding P. Attenuation of human lung mast cell degranulation by bronchial epithelium. Allergy 2006; 61:569-75. 143. Cutz E, Levison H, Cooper DM. Ultrastructure of airways in children with asthma. Histopathology 1978; 2:407-21.

266

MAST CELL BIOLOGY

144. Wang SW, Oh CK, Cho SH et al. Amphiregulin expression in human mast cells and its effect on the    #$ $   >   @ $ _••– _—”^€’ 145. Enomoto Y, Orihara K, Takamasu T et al. Tissue remodeling induced by hypersecreted epidermal growth factor and amphiregulin in the airway after an acute asthma attack. J Allergy Clin Immunol 2009; 124:913-20 e1-7. 146. Kalesnikoff J, Huber M, Lam V et al. Monomeric IgE stimulates signaling pathways in mast cells that lead to cytokine production and cell survival. Immunity 2001; 14:801-11. 147. Kitaura J, Song J, Tsai M et al. Evidence that IgE molecules mediate a spectrum of effects on mast cell survival and activation via aggregation of the FcepsilonRI. Proc Natl Acad Sci USA 2003; 100:12911-6. 148. Pandey V, Mihara S, Fensome-Green A et al. Monomeric IgE stimulates NFAT translocation into the nucleus, a rise in cytosol Ca2   $    ${  # $$   # leukemia-2H3 mast cell line. J Immunol 2004; 172:4048-58. ’€ ˆ< * `   *<    = ^$     $   degranulation in RBL-2H3 mast cells. Am J Physiol Cell Physiol 2004; 286:C256-63. 150. Cruse G, Kaur D, Yang W et al. Activation of human lung mast cells by monomeric immunoglobulin E. Eur Respir J 2005; 25:858-63. 151. Cruse G, Cockerill S, Bradding P. IgE alone promotes human lung mast cell survival through the autocrine production of IL-6. BMC Immunol 2008; 9:2. 152. Burrows B, Martinez FD, Halonen M et al. Association of asthma with serum IgE levels and skin-test reactivity to allergens. N Engl J Med 1989; 320:271-7. 153. Sears MR, Burrows B, Flannery EM et al. Relation between airway responsiveness and serum IgE in children with asthma and in apparently normal children. N Engl J Med 1991; 325:1067-71. 154. Sunyer J, Anto JM, Sabria J et al. Relationship between serum IgE and airway responsiveness in adults with asthma. J Allergy Clin Immunol 1995; 95:699-706. 155. Sunyer J, Anto JM, Castellsague J et al. Total serum IgE is associated with asthma independently of   = ! *# # $  # =$  $  # =$ ‚ > €€“– €——•^’ 156. Djukanovic R, Wilson SJ, K Kraft M et al. Effects of treatment with anti-immunoglobulin E antibody }$   % {    #  > ‚ @  @   _••’– ”•—Œ^€Œ 157. Columbo M, Horowitz EM, Botana LM et al. The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils. J Immunol 1992; 149:599-608. 158. Al-Muhsen SZ, Shablovsky G, Olivenstein R et al. The expression of stem cell factor and c-kit receptor in human asthmatic airways. Clin Exp Allergy 2004; 34:911-6. 159. Da Silva CA, Blay F, Israel-Biet D et al. Effect of glucocorticoids on stem cell factor expression in human asthmatic bronchi. Clin Exp Allergy 2006; 36:317-24. 160. Chong LK, Suvarna K, Chess-Williams R et al. Desensitization of beta2-adrenoceptor-mediated responses by short-acting beta2-adrenoceptor agonists in human lung mast cells. Br J Pharmacol 2003; 138:512-20. 161. Giannini D, Carletti A, Dente FL et al. Tolerance to the protective effect of salmeterol on allergen challenge. Chest 1996; 110:1452-7. 162. Swystun VA, Gordon JR, Davis EB et al. Mast cell tryptase release and asthmatic responses to allergen increase with regular use of salbutamol. J Allergy Clin Immunol 2000; 106:57-64. 163. Taylor DR, Sears MR, Herbison GP et al. Regular inhaled beta agonist in asthma: effects on exacerbations and lung function. Thorax 1993; 48:134-8. 164. Taylor DR, Town GI, Herbison GP et al. Asthma control during long-term treatment with regular inhaled salbutamol and salmeterol. Thorax 1998; 53:744-52. 165. Cruse G, Yang W, Duffy SM et al. Counterregulation of beta(2)-adrenoceptor function in human mast cells by stem cell factor. J Allergy Clin Immunol 2010; 125:257-63. 166. Elliot JG, Abramson MJ, Drummer OH et al. Time to death and mast cell degranulation in fatal asthma. Respirology 2009; 14:808-13. 167. Marc MM, Korosec P, Kosnik M et al. Complement factors c3a, c4a and c5a in chronic obstructive pulmonary disease and asthma. Am J Respir Cell Mol Biol 2004; 31:216-9. “— ~ š š#  ` #     ‚$     % { # $# *^<  ’^      $ \  ~   ' _••“– •Œ__—“^€ 169. Murakami D, Yamada H, Yajima T et al. Lipopolysaccharide inhalation exacerbates allergic airway {  !      *#_  @ ="   _••”– Œ”ŒŒ€^’” ”• ˆ<$$   #%<$  > * `         "      #$ mast cells mediated by Toll-like receptor 4 and FcepsilonRI. Blood 2003; 102:2547-54. 171. Kato A, Favoreto S, Jr., Avila PC et al. TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells. J Immunol 2007; 179:1080-7.

MAST CELLS IN LUNG INFLAMMATION

267

172. Allakhverdi Z, Comeau MR, Jessup HK K et al. Thymic stromal lymphopoietin is released by human #        $  {   !   > =" Med 2007; 204:253-8. 173. Zhou B, Comeau MR, De Smedt T et al. Thymic stromal lymphopoietin as a key initiator of allergic  % {   ~ $ _••– “•’”^Œ ”’  * & š` $  ˆ   *ƒ\^!    $  {  * #  2 cell response through OX40 ligand. J Exp Med 2005; 202:1213-23. 175. Seshasayee D, Lee WP, Zhou M et al. In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin  !  { > @ ! _••”– ”Œ—“—^”— 176. Miyata M, Hatsushika K, Ando T et al. Mast cell regulation of epithelial TSLP expression plays an important role in the development of allergic rhinitis. Eur J Immunol 2008; 38:1487-92. 177. Ying S, O’Connor B, Ratoff J et al. Expression and cellular provenance of thymic stromal lymphopoietin and chemokines in patients with severe asthma and chronic obstructive pulmonary disease. J Immunol 2008; 181:2790-8. 178. Ying S, O’Connor B, Ratoff J et al. Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity. J Immunol 2005; 174:8183-90. 179. Redegeld FA, van der Heijden MW, Kool M et al. Immunoglobulin-free light chains elicit immediate hypersensitivity-like responses. Nat Med 2002; 8:694-701. 180. Kraneveld K AD, Kool M, van Houwelingen AH et al. Elicitation of allergic asthma by immunoglobulin free light chains. Proc Natl Acad Sci USA 2005; 102:1578-83. 181. Huber M, Helgason CD, Damen JE et al. The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation. Proc Natl Acad Sci USA 1998; 95:11330-5. 182. Vonakis BM, Gibbons S, Jr., Sora R et al. Src homology 2 domain-containing inositol 5v phosphatase is negatively associated with histamine release to human recombinant histamine-releasing factor in human basophils. J Allergy Clin Immunol 2001; 108:822-31. 183. Dudler T, Machado DC, Kolbe L et al. A link between catalytic activity, IgE-independent mast cell activation and allergenicity of bee venom phospholipase A2. J Immunol 1995; 155:2605-13. 184. Machado DC, Horton D, Harrop R et al. Potential allergens stimulate the release of mediators of the            #   } %# ^  = Eur J Immunol 1996; 26:2972-80. 185. Huels C, Germann T, Goedert S et al. Co-activation of naive CD4 T-cells and bone marrow-derived mast cells results in the development of Th2 cells. Int Immunol 1995; 7:525-32. 186. Pawankar R, Okuda M, Yssel H et al. Nasal mast cells in perennial allergic rhinitics exhibit increased expression of the Fc epsilonRI, CD40L, IL-4 and IL-13 and can induce IgE synthesis in B cells. J Clin Invest 1997; 99:1492-9. —” & @   >      % !  $   #  {  an asthma model in mice. J Exp Med 2000; 192:455-62. 188. Taube C, Wei X, Swasey CH et al. Mast cells, Fc epsilon RI and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen exposure in the absence of adjuvant. J Immunol 2004; 172:6398-406. 189. Yu M, Tsai M, Tam SY et al. Mast cells can promote the development of multiple features of chronic asthma in mice. J Clin Invest 2006; 116:1633-41. €• & † `$  &  \>   @$      #   #   impairs murine airway allergic responses. J Immunol 2006; 177:5779-84. 191. Cyphert JM, Kovarova M, Allen IC et al. Cooperation between mast cells and neurons is essential for antigen-mediated bronchoconstriction. J Immunol 2009; 182:7430-9. 192. van Houwelingen AH, Kool M, de Jager SC et al. Mast cell-derived TNF-alpha primes sensory nerve endings in a pulmonary hypersensitivity reaction. J Immunol 2002; 168:5297-302. 193. Wessler I, Holper B, Kortsik C et al. Dysfunctional inhibitory muscarinic receptors mediate enhanced histamine release in isolated human bronchi. Life Sci 2007; 80:2294-7. 194. Strieter RM. Pathogenesis and natural history of usual interstitial pneumonia: the whole story or the last chapter of a long novel. Chest 2005; 128:526S-32S. €  #  ‚<#           #   $   "  $  $      $##   $ # } $  > \# 1994; 145:114-25. €“ `  †= %  @  †         #    #$ $       ! '  $$      $  > \# 1992; 166:303-10. €” @ *† *    # †   † #! ! {$ # !    $  Am Rev Respir Dis 1988; 138:1604-8.

268

MAST CELL BIOLOGY

198. Gonzalez NC, Allen J, Schmidt EJ et al. Role of the renin-angiotensin system in the systemic microvascular {  ! #"  > \# `  @  \# _••”– _€_`__—^€’ €€ $ ‚ \ž  

 <    \#     $  $$    J Immunol 1983; 130:341-9. _•• †$< ‚> ~ ~ƒ `  #    < $     %#        \   =" †  €”Œ– ’’€_€^ŒŒ 201. Jordana M, Befus AD, Newhouse MT et al. Effect of histamine on proliferation of normal human adult $   *# " €——– ’Œ_^— 202. Feghali CA, Bost KL, Boulware DW et al. Human recombinant interleukin-4 induces proliferation and  $<^“  $  $$  #$ <   @ $ $# €€_– “Œ—_^” 203. Sugarman BJ, Aggarwal BB, Hass PE et al. Recombinant human tumor necrosis factor-alpha: effects on proliferation of normal and transformed cells in vitro. Science 1985; 230:943-5. 204. Hetzel M, Bachem M, Anders D et al. Different effects of growth factors on proliferation and matrix  $       #$ $   ƒ$ _••– —Œ__^Œ” _• * $  $  † =  ~   `  ^ #  #$      Arch Dermatol Res 1997; 289:194-203. _•“ * $   #  † < =†   `$   $       #  cell-cell adhesion and action of IL-4. J Immunol 1998; 160:5053-7. 207. Bradding P, Feather IH, Wilson S et al. Immunolocalization of cytokines in the nasal mucosa of normal and perennial rhinitic subjects. The mast cell as a source of IL-4, IL-5 and IL-6 in human allergic mucosal { > $ €€Œ– Œ—Œ^“ 208. Postlethwaite AE, Seyer JM. Fibroblast chemotaxis induction by human recombinant interleukin-4.    #    % #      ‹$ 70-88 and 89-122. J Clin Invest 1991; 87:2147-52. _•€ \#% = `   `   `$   #} ! !  " $ matrix proteins in response to interleukin 4. J Clin Invest 1992; 90:1479-85. _•  %}  @# > ƒ$          %#   # ^#    #  #  %# $     %#   \  ~   ' €—’– 81:6963-7. 211. Yayon A, Klagsbrun M, Esko JD et al. Cell surface, heparin-like molecules are required for binding of     %#    ##     @ €€– “’—’^— __ < >  $   >    # ^   ¢    %# factor—is stored within basement membrane. Am J Pathol 1988; 130:393-400. _Œ <   \   ` ‚       $ #$ $      ! protease-activated receptor-2. Am J Physiol Lung Cell Mol Physiol 2000; 278:L193-201. 214. Goto T, Befus D, Low R et al. Mast cell heterogeneity and hyperplasia in bleomycin-induced pulmonary

     ‚! ‚  €—’– Œ•”€”^—•_ _ &  &  ˆ# *      #  !  $     ionizing irradiation. Ultrastructural and histochemical studies. Lab Invest 1974; 31:555-67. 216. Wagner MM, Edwards RE, Moncrieff CB et al. Mast cells and inhalation of asbestos in rats. Thorax 1984; 39:539-44. 217. Suzuki N, Horiuchi T, Ohta K et al. Mast cells are essential for the full development of silica-induced $  {  $ %#  ^    > ‚ @  † €€Œ– €’”^—Œ 218. Assaad AM, Kawut SM, Arcasoy SM et al. Platelet-derived growth factor is increased in pulmonary capillary hemangiomatosis. Chest 2007; 131:850-5. _€ ˆ§†  > #   @#  $ !  {  # $   Br J Cancer 2001; 85:473-83. 220. Ribatti D, Crivellato E. The controversial role of mast cells in tumor growth. Int Rev Cell Mol Biol 2009; 275:89-131. 221. Theoharides TC, Conti P. Mast cells: the Jekyll and Hyde of tumor growth. Trends Immunol 2004; 25:235-41. 222. Takanami I, Takeuchi K, Naruke M. Mast cell density is associated with angiogenesis and poor prognosis in pulmonary adenocarcinoma. Cancer 2000; 88:2686-92. 223. Imada A, Shijubo N, Kojima H et al. Mast cells correlate with angiogenesis and poor outcome in stage I lung adenocarcinoma. Eur Respir J 2000; 15:1087-93. 224. Nagata M, Shijubo N, Walls AF et al. Chymase-positive mast cells in small sized adenocarcinoma of the lung. Virchows Arch 2003; 443:565-73. 225. Ribatti D, Vacca A, Nico B et al. Bone marrow angiogenesis and mast cell density increase simultaneously with progression of human multiple myeloma. Br J Cancer 1999; 79:451-5. __“ @$ ƒ  ‚ && †     {    $^ $  $  squamous epithelial carcinogenesis. Genes Dev 1999; 13:1382-97.

MAST CELLS IN LUNG INFLAMMATION

269

__” ‚$#  †} š š ‚   *# {$          &”“  ! carcinoma cells. J Pharmacol Sci 2008; 106:208-18. 228. Zudaire E, Martinez A, Garayoa M et al. Adrenomedullin is a cross-talk molecule that regulates tumor and mast cell function during human carcinogenesis. Am J Pathol 2006; 168:280-91. 229. Tomita M, Matsuzaki Y, Onitsuka T. Effect of mast cells on tumor angiogenesis in lung cancer. Ann Thorac Surg 2000; 69:1686-90. 230. Kojima H, Shijubo N, Abe S. Thymidine phosphorylase and vascular endothelial growth factor in patients with Stage I lung adenocarcinoma. Cancer 2002; 94:1083-93. 231. Ibaraki T, Muramatsu M, Takai S et al. The relationship of tryptase- and chymase-positive mast cells to angiogenesis in stage I nonsmall cell lung cancer. Eur J Cardiothorac Surg 2005; 28:617-21. _Œ_ $ = ˆ ' \< †@   *#    # %    $$ angiogenesis in nonsmall cell lung carcinoma. J Int Med Res 2008; 36:88-95. 233. Carlini MJ, Dalurzo MC, Lastiri JM et al. Mast cell phenotypes and microvessels in nonsmall cell lung        `$ \# _••€ 234. Tataroglu C, Kargi A, Ozkal S et al. Association of macrophages, mast cells and eosinophil leukocytes with angiogenesis and tumor stage in nonsmall cell lung carcinomas (NSCLC). Lung Cancer 2004; 43:47-54. _Œ ‘ ` #‘$ ~ š   @    !$ #  %#  ^@  vascular endothelial growth factor receptor 3 in patients with T1 lung adenocarcinoma. Cancer 2005; 104:1668-77. 236. Tomita M, Matsuzaki Y, Onitsuka T. Correlation between mast cells and survival rates in patients with pulmonary adenocarcinoma. Lung Cancer 1999; 26:103-8. 237. Welsh TJ, Green RH, Richardson D et al. Macrophage and mast-cell invasion of tumor cell islets confers a marked survival advantage in nonsmall-cell lung cancer. J Clin Oncol 2005; 23:8959-67. 238. Tomita M, Matsuzaki Y, Edagawa M et al. Distribution of Mast Cells in Mediastinal Lymph Nodes from Lung Cancer Patients. World J Surg Oncol 2003; 1:25. _Œ€ % ˆ #  $    \       #  @—( ) T-Cells in cancer         $ !!   ž   $   @ _••—– ŒŒ—”^€

CHAPTER R 14

& $ % +( $%$+ AND CANCER  ‚|,1 and Enrico Crivellato2 1

Department of Human Anatomy and Histology, University of Bari Medical School, Piazza Giulio Cesare Bari, Italy; Department of Medical and Morphological Reseach, Section of Anatomy, University of Udine Medical School, Udine, Italy    {  `&€ &  & 2

Abstract:

  ˜ @™ %      \$ =# #1 in his doctoral thesis. MCs have long been implicated in the pathogenesis of allergic reactions and certain protective       $   {      %## often include plentiful MCs, the question as to the possible contribution of MCs to $ ! #   !     # #  #   involvement of MCs in tumor biology and tumor fate will be considered, with particular emphasis on the capacity of these cells to stimulate tumor growth by promoting angiogenesis and lymphangiogenesis. Data from experimental carcinogenesis and from different tumor settings in human pathology will be summarized. Information to be presented will suggest that MCs may serve as a novel therapeutic target for cancer treatment.

INTRODUCTION

  ˜ @™   !  $^#     %## %   described by Paul Ehrlich1 in his doctoral thesis.2 MCs have long been implicated in the pathogenesis of allergic reactions and certain protective responses to parasites. They have been linked to various cell-mediated immune reactions, being found in tissues from multiple disease sites and as a component of the host reaction to bacterial, fungal and viral infections. They have also been shown to participate in angiogenesis and tissue repair processes after injury.3,4

Mast Cell Biology: Contemporary and Emerging Topics       and Dean D. Metcalfe. ©2011 Landes Bioscience and Springer Science+Business Media. 270

& $ % +( $%$+ % %$*

271

*#      $ < % #  {   #   }   $   {      which often include plentiful MCs, the question as to the possible contribution of MCs to tumor development has been emerging. A major point linking MCs to cancer is the well recognized capacity of these cells to synthesize and release potent angiogenic compounds. Although some evidence suggests that MCs can promote tumorigenesis and tumor progression, there is some clinical evidence, as well as experimental tumor models, where MCs seem to have functions that favour the host. Thus, the involvement of MCs in tumor development is complex and far from being settled. & % " #   !!  @  $   $ fate, with particular emphasis on the capacity of these cells to stimulate tumor growth by promoting angiogenesis and lymphangiogenesis. Then, data from experimental carcinogenesis and from different tumor settings in human pathology will be reviewed. The last section will be dedicated to drugs affecting MC number.

MAST CELLS IN EXPERIMENTAL CARCINOGENESIS Involvement of MCs in experimental carcinogenesis has increasingly attracted the interest of researchers. Development of squamous cell carcinoma in a HPV (human papilloma virus) 16-infected transgenic mouse model of epithelial carcinogenesis has provided experimental evidence in favour of an early participation of MCs in tumor growth and angiogenesis.5 Accumulation of MCs was observed at the hyperplastic and dysplastic stages and in the invasive edge of tumor growth. They were typically localized around capillaries and epithelial basement membrane, where MC degranulation resulted in #   # @^       # @ %   %# # $       @  !   "   (MMP)-9 coincided with the angiogenic switch in premalignant lesions. Addition of #  % $   $   # %# $ with a hyperplastic skin sample in vitro, while tryptase was shown to play a prominant role in tissue remodelling. Gounaris et al6 developed a transgenic mouse model over-expressing an inducible, stabilized `–catenin in intestinal enterocytes, as well as the APC6468 as an independent #       #  !  %      by CD34 @–  #    #    # ! and mucosal type MCs in developing tumors. The latter MC type was prevalent at the site of newly developing polyps, while the former was prevalent in the stroma at later stages of tumor development. Treatment with antitumor necrosis factor alpha (TNF_  $  %  $   %#    @    Reconstitution of Wsh/Wsh @^   %# @Œ’
272

MAST CELL BIOLOGY

!  $   #     $  %#  #      with the expansion of islet tumors. Macrophages and neutrophils were also recruited after the onset of tumor angiogenesis. Treatment of established `-cell tumors with disodium cromoglycate (cromolyn), an inhibitor of MC degranulation, rapidly triggered hypoxia and cell death of tumor and endothelial cells. The authors concluded that MCs are necessary for tumor angiogenesis and growth; and suggested that inhibition of MC function might prove to be therapeutically useful in restraining the growth of pancreatic cancer. In an in vivo transplantation assay in nude mice, angiopoietin-1 (Ang-1) secreted by primary murine MCs promoted marked neovascularization.8 The MCs studied were bone marrow mast cells (BMMCs) from C57BL/6 mice and two mouse plasmocytoma cell lines. Greater angiogenesis was demonstrated when BMMCs were transplanted into mice together with plasmocytoma cells, than when each of the cell types was transplanted  *# $  ^^$ }    $ #  %#  plasmocytoma containing MC. Overall, these data demonstrate that MC-derived Ang-1 promotes growth of plasmocytomas by stimulating neovascularization. Hart et al,9 using W/Wv mice, demonstrated a direct correlation between MC density in the dermis and susceptibility to ultraviolet-B-induced systemic immunosuppression. These mice, which are homozygous for the W (white spotting) mutation and therefore !  @     $ !  $ !^$ $$  $

 ‘ %# @  $    #   9 The W locus encodes the KIT tyrosine kinase receptor that binds stem cell factor (SCF).10 The MC products involved in ultraviolet-induced immunosuppression are believed to be TNF_ and histamine.11 These data lend support for a crucial role of MCs in early carcinogenesis in different experimental settings.

MAST CELL RECRUITMENT TO TUMOR ‚  $    @    $   {  # $   # $  !  @#  { $ both proliferation of resident tissue MCs and recruitment of MCs and their precursors from outside the lesion.12,13 During tumor development, circulating MC precursors migrate into the neoplastic mass and form one of the major stromal cell populations. Recruitment and !  @  # $        $ ^ !   factor (SCF) through its receptor KIT on MCs.14 In a hepatocarcinoma model, MCs failed to migrate into SCF-knockdown tumors and anti-KIT antibodies abolished the migration of MCs into tumors, leading to decreased tumor growth. Low concentrations of SCF   $ # #    @  #   !

\^€  the local environment. Higher SCF concentrations promoted activation of recruited MCs  # $ " %## %  $    ^{    $ interleukin (IL)-6, TNF-_, vascular endothelial growth factor (VEGF), Cox-2, i-NOS and CCL-2.14 The intervention of SCF-activated MCs in the tumor microenvironment increased the transcription of the IL-17 gene and the number of IL-17-producing cells in the tumor   $  ƒ^”     $ # $ {   # $# #  $  ƒ^€ %$     @  #   {15 TNF-_  #  ^{      @      # activity of NK-gB and AP-1 in tumor cells.14 NK-gB and AP-1 may, in turn, favour the proliferation of tumor cells by inducing the expression of cyclines, the survive of tumor

& $ % +( $%$+ % %$*

273

cells by the blockade of apoptosis and the invasiveness of tumor cells by inducing the production of macrophage migration inhibitory factor (MIF) and EMMPRIN.16,17

MAST CELLS AND IMMUNE TOLERA R NCE A new paradigm of immunological participation for MCs has recently been proposed to explain their involvement in the dynamics of tumor development and progression. Although immune surveillance works at an early stage of tumorigenesis, established tumors primarily induce immune tolerance by creating sites of immune privilege and by inducing a shift of the immune balance from activation to tolerance.18,19 MCs have recently been proposed to be mechanistically involved in the negative modulation of immune surveillance in the tumor microenvironment (Fig. 1). MCs have thus been found to play a critical role in the suppression of immune reactions.20 These cells not only produce inhibitory cytokines, such as IL-10,21 but also are essential in promoting the immune tolerance mediated by regulatory T (Treg) cells. Indeed, MCs serve as enforcers for Treg cells, turning down the immune system’s reaction to a skin allograft, possibly through IL-10 secretion.22 Ultraviolet-B irradiation which represents the most important skin immunesuppressor and initiator of cutaneous malignancies, activates MCs.23,24 Upon

Figure 1. *# $^$ !   @  # $ !  @    # $ stroma favor expansion and activation of regulatory T cells which, in turn, stimulate immune tolerance and tumor promotion. SCF-mediated recruitment of MCs by tumor cells leads to MC activation and release of immunosuppressive and tumor-promoting molecules such as IL-10, histamine and TNF-_. Upon activation, regulatory T cells release SCF and IL-9, which expand the MC population. MCs serve as enforcers for regulatory T cells, possibly by MHC-mediated activation. Abbreviations: T reg cell, regulatory T-cell; SCF, stem cell factor; TNF-_, tumor necrosis factor-_; MHC, major histocompatibility complex; IL-9, IL-10, IL-17, interleukin-9, -10, -17.

274

MAST CELL BIOLOGY

irradiation of the skin, trans-urocanic acid in the epidermis isomerizes to cis-urocanic  %## $ $     $  ^   *# $  in turn, trigger secretion of histamine, TNF-_ and other mediators from MCs, leading to suppression of the cellular immune system.11,25 ' @^  &&v mice, a direct correlation has been demonstrated between MC density in the dermis and susceptibility to ultraviolet-B-induced systemic immunosuppression.9 In a skin transplantation model of      # $ @ %   $      @^  C57BL/6-Kit W-sh/W-sh mice showed an inability to induce tolerance.22 Activated Treg cells in the tolerant tissue produced high levels of IL-9, a cytokine which seems important in MC recruitment, growth and activation. This cytokine appears to be a crucial factor in mediating regional tolerance, as neutralization of IL-9 greatly accelerates allograft rejection in tolerant mice.22 In a mouse model of tumorigenesis, SCF-activated MCs exacerbated immunosuppression in the tumor microenvironment.14 MCs were shown to promote a decrease in mRN R A for the immune activating factor IL-2, while inducing an increase in mRN R As for the immune suppressor factors IL-10, transforming growth factor beta (TGF-` and forkhead box p 3 (Foxp3) in the tumor. The percentage of Treg cells in the total T-cell number in the tumor mass was also increased because CD4 CD25< T cells can be converted into CD4 CD25 regulatory T cells by TGF-` induced expression of Foxp3.26 # #   #  #    MC-driven Treg cell expansion might be linked to the lipopolysaccharide (LPS) and interferon (IFN)-a-induced expression of MHC class II on MCs. The expression of MHC-II grants MCs the ability to process and present antigens directly to T cells. In  @    " ^  *   ! ´! *  %## may help explain allograft tolerance induction by MCs.27 MCs are poor stimulators of naïve T cells in vitro. However, one consequence of MHC-II expression on MCs may be to activate regulatory Treg cells and thus dampen the immune response. Activation of Treg cells by MCs may also thus contribute to the protective effect of MCs on skin allografts.22,27 A recent publication supports the role of MC-Treg cell interactions in promoting immunosuppression and tumor development. The density of peritumoral MCs and Treg cells was evaluated to predict the prognosis of hepatocellular carcinoma.28 It was found that higher peritumoral MC numbers were associated with poorer clinical outcome. Peritumoral Treg cells were positively correlated with MC density and inversely related to hepatocellular carcinoma outcomes. Further, MC number in combination with Treg cell number could predict prognostic outcomes more effectively than MC numbers alone. Thus, MCs and Treg cells may cooperate with each other in hepatocellular carcinoma to result in a poorer prognosis.28 In conclusion, the pivotal role of MCs in the induction of an acquired condition of immune tolerance appears to be well established in various experimental settings. The secretion of immunosuppressive mediators by MCs seems to be critical for sustaining tolerance.

MAST CELLS AND TUMOR GROWTH IN HUMANS As noted, MCs accumulate at sites of tumor growth in response to numerous chemoattractants, in particular SCF secreted by tumor cells.14,29 MCs are endowed with a vast array of mediators, some of which have promoting and others, inhibitory effects on malignancies.30 Degradation of the extracellular matrix (ECM) plays a critical role in tumor expansion and metastasis. Dabbous et al31 showed that MC degranulation is associated

& $ % +( $%$+ % %$*

275

with disruption and lysis of the tumor ECM, either directly through the action of their }    # $# $  #  !    macrophages and tumor cells.32,33 Tryptase activates latent MMP and plasminogen activator, which, in turn, degrade the ECM.32 MCs cause apoptosis in various target cells34 and $ # $$  $  {    !!35 Heparin enhances both the activity and production of collagenase in vitro and release plasminogen activator from endothelial cells.36,37 MCs are a major source of histamine, %## $ $ $     # $# `      #$ malignant carcinoma, while suppressing the immune system through H2 receptors.38 High histamine concentrations inhibit human primary melanoma-cell proliferation, presumably by acting through H1 receptors, an action enhanced by IL-2. Low amounts of histamine acting through H2 receptors increased this cell proliferation.39 The incidence of metastases, as well as the appearance of tumors, correlates inversely with the tissue histamine level.40 MCs may also exert direct mitogenic effect on tumor cells through     "  ^ !    %#  ˜ ™^_ and IL-8 are directly mitogenic to melanocytes and melanoma cells.41,42 ` "   <  $  #   @  # {       @      $  % $ to be much higher than in control subjects43 and MC accumulation is also increased in well-differentiated gastric cancers when compared with controls. These observations $    @  # # % {    `%!  poorly-differentiated gastric adenocarcinomas showed lower MC density than well-differentiated adenocarcinomas.43 MCs have also been detected in bilateral multiple spindle cell lipomas of the tongue.44 It has similarly been suggested that MCs may be       $   #     $     the skin.45 †     @ %      %#    $    %    #  @ #!  $    numbers at the periphery and in between the tumor islands of basal cell carcinomas.46 In addition, MCs in basal cell carcinoma have been found to express VEGF, IL-8 and CCL5 (RAN A TES), suggesting an active MC involvement in the tumor microenvironment.47    $  @ #    # $      telangectasia macularis eruptiva perstans lesions,48 in a case of conjunctival myxoma,49 and in synovial sarcoma.50

CORRELATION WITH PROGNOSIS   $  # $     @ #  #%    %# #  $                ! increase in MC number associated with a increasingly poor prognosis has been shown during the progression of human melanoma and oral squamous carcinoma.51,52 Rojas et al53 demonstrated a higher number of MCs in squamous cell carcinoma of the lip, as compared to normal lip. Sharma et al54 reported a higher number of MCs in nodular sclerotic-type Hodgkin’s lymphoma (HL) than in other types of HL, and the number of MCs was higher      #  $     55,56 observed a worse prognosis for a nodular sclerosing HL exhibiting a high MC number. Fukushima et al57 demonstrated an   $  @       $   †^ # ˜ƒ@@ƒ™ lymph nodes. The greatest number of MCs among T-cell lymphomas was observed in

276

MAST CELL BIOLOGY

angioimmunoblastic T-cell lymphoma.58 Dave et al59 utilized gene array to study the # %        "      $ that the length of survival among patients with follicular lymphoma correlated with the molecular features of nonmalignant immune cells present within the tumor at the time of diagnosis. One of the genes observed to correlate most negatively with survival was microphthalmia-associated transcription factor (MITF), a transcription factor which has been found to be highly expressed in MCs and to play a critical role in the regulation  !  < @^  60 Tournilhac et al61 demonstrated that MCs may support tumor cell expansion in Waldenstrom’s macroglobulinemia through constitutive CD154-CD40 signalling. Moreover, the use of a CD154-CD40 signal inhibitor partially inhibited MC mediated bone marrow lymphoplasmacytic proliferation and/or tumor colony formation. Nonomura et al62 demonstrated in prostate cancer that MC counts were higher around cancer foci in patients with higher Gleason scores than in those with low Gleason scores. The MC number correlated with clinical stage and multivariate analysis revealed # @ $ %        †   ##  $  @ density was associated with favourable tumor characteristics and a good prognosis in prostate cancer.63 *#        %#    @  # immunological host-defense reaction to prostate cancer. Gomes et al64 detected an increased MC density in actinic cheilitis and in squamous cell carcinoma of the lip compared to normal oral mucosa, suggesting a role for MCs in # !      #    %     ƒ Costa et al.65 In another study, cyclooxygenase (COX)-2 over-expression, tryptase-positive MCs and proteinase-activated receptor (PAR)-2 were investigated in normal lip biopsies and in lip biopsies with actinic cheilitis.65 MC-derived tryptase was found to induce COX-2 expression by the cleavage of PAR-2. Increased epithelial co-expression of COX-2 and PAR-2, as well as an increase in the subepithelial density of tryptase-positive MCs were found in actinic cheilitis compared to normal lip. COX-2 overexpression was found to be        #      %# #  ^! MCs and PAR-2 expression. The authors concluded that tryptase may contribute to COX-2 up-regulation by epithelial PAR-2 activation during early lip carcinogenesis.66 However, the number of tryptase- and KIT-positive MCs was found to be decreased in oral squamous cell carcinoma biopsies by Oliveira-Neto et al.67 Aaltomaa et al68 found a positive correlation between survival and increased MC number in a study of 187 breast cancer biopsies. Dabiri et al69 analyzed the correlation between MC number in breast cancer and patients’ prognosis in a study of 438 patients. They found a strong correlation between the presence of MCs and a favourable prognosis. Perivascular tumor-associated MCs in mammary adenocarcinoma could secrete several cytokines and proteolytic enzymes that could be detrimental to the tumor cells. For instance, IL-4, which binds to IL-4 receptors expressed by human breast carcinoma cells, could lead to apoptosis in breast cancer.70 However, the histamine content of human breast cancer tissue is much higher than adjacent normal tissue and could act as a local immunosuppressant.71 Moreover, the mean level of serum tryptase in women with breast cancer is three-times higher than in healthy women.72 Kankkunen et al73 similarly observed # #      @ $      ! $    due to tryptase-containing MCs. In benign lesions, the number of MCs exhibiting tryptase activity was similar to that of chymase containing MCs. Malignant tumors, however, had 2-3 times more tryptase-containing, than chymase-containing, MCs and the tryptase

& $ % +( $%$+ % %$*

277

! %   ## #     !     tryptase-containing MCs were concentrated at the tumor edge, whereas chymase-containing MCs were not increased in this area. Breast cancer patients with metastases in the axillary nodes revealed greater numbers of MCs in all nodes examined, compared with patients without metastasis.74 Della Rovere et al,75 however, argued that MCs may play a protective       *# $ # #  %  ##      @     $     #! ## #    %# respect to both hypo-hormonal cancers in the same location and to controls. In another study, stromal MCs were found to correlate to low grade invasive breast carcinoma and estrogen receptor positivity.76 Welsh et al77 analyzed the presence of MCs in the tumor stroma of 175 patients with surgical resected non small cell lung carcinoma. They demonstrated that both macrophage  @     # $  %  %#   <    ^ survival, independently of other favourable prognostic factors including stage. Chan et al78 studied samples of ovarian cancer from 44 patients and demonstrated that patients with tumors with higher microvascular density had a higher mean survival compared to those with tumors with low MC density or low microvessel density. Cinel et al79 showed a     % ## @   #      !          # $  $     ^! MC after IL-2 preoperative induction therapy predicted an improved clinical outcome in patients with malignant pleural mesothelioma. This highlights the critical role of the  {    #    80

ANGIOGENESIS FACTORS STORED IN MAST CELLS MCs synthesize and store large amounts of molecular compounds exerting remarkable activity upon different aspects of angiogenesis. Heparin stimulates endothelial cell proliferation and migration in vitro.81,82 In vivo, however, it has been reported to stimulate,83-85 inhibit 85-87 or have no effect88,89 on angiogenesis. These differences seem to be related to the molecular size and degree of sulphation of the heparin employed. Heparin acts as a $    # %^   ^_  90 displacing FGF-2 in the biologically active form and allowing FGF-2 rapid interaction with endothelial cells.91 Histamine has an angiogenic effect through both H1 and H2 receptors.92 It may also increase the permeability of newly formed microvessels during tumor angiogenesis  #   # <             $        !!93 MC synthesize and store large amounts of MMP-2, MMP-9 and serine-proteinases of two subclasses: tryptase and chymase.94-96 Given the ability of MMP-2 and MMP-9    * ž ž ž      97 the major components of the interstitial stroma and subendothelial basement membrane, these facts suggest that MCs may contribute to the in situ progression of invasive and metastatic solid tumors.97 Tryptase and chymase are involved in angiogenesis after their release from activated MC granules. Their proteolytic activities degrade ECM components, release matrix-associated growth factors,98 and act indirectly by activating latent MMP99 and plasminogen activators.32 Blair et al100 have demonstrated the angiogenic potential of tryptase in vitro and its important role in neovascularization. Muramatsu et al101,102 used the hamster sponge-implant model to show that angiogenesis is induced by angiotensin

278

MAST CELL BIOLOGY

II and inhibited by chymase inhibitors. These observations suggest that MC-derived chymase is an important mediator of MC-dependent angiogenesis. MCs release polypeptide growth factors including FGF-2, VEGF, TNF-_, TGF-` and IL-8. These cytokines are involved in normal, as well as tumor-associated angiogenesis. The spectrum of cytokines expressed appears to vary depending on the maturity state of the MC and the tissue of residence. Qu et al103 demonstrated that FGF-2 is localized in the cytoplasmic and extruded granules of MCs in several human tissues. Grutzkan et al104 demonstrated the expression of VEGF in the human MC line HMC-1 and in human skin MCs. Nerve growth factor (NGF), also contained in MC secretory granules, induces endothelial cell proliferation in vitro and angiogenesis in vivo in the chick embryo chorioallantoic membrane (CAM) assay.105,106

MAST CELLS IN EXPERIMENTAL TUMOR ANGIOGENESIS Kessler et al107 demonstrated that tumor angiogenesis factor (TAF) elicited a vasoproliferative response when placed upon the CAM of the chick embryo. The action of TAF might depend upon recruitment of MCs. Poole and Zetter108 demonstrated that rat peritoneal MCs migrate in response to conditioned medium from several tumor cell lines. The active chemoattractant(s) in this conditioned medium appeared to be peptide(s) with a molecular weight of 300-1,000. They proposed that the chemoattraction of MCs by tumor-derived peptides may be an important early event in tumor neovascularization. Starkey et al109 investigated the role of host MCs in tumor-associated angiogenesis    #     @^  &&v mice and

@^$  / litter mates to subcutaneously growing B16-BL6 tumors. The response was slower and initially less intense in W/Wv mice. Fewer W/Wv mice compared to /   ! $ $  †  %   # @   restored the incidence of haematogenous metastases to approach that of / mice. These results demonstrate a role for MCs in vivo during tumor angiogenesis and also suggest a role for host MCs in hematogeneous metastases. We have demonstrated that isolated MCs or their secretory granules, but not degranulated MC, induce an angiogenic response in the CAM assay.110 Addition of anti-FGF-2 or anti-VEGF antibodies reduced the angiogenic response of both MCs and their secretory granules by 50% and 30% respectively. These data provide evidence that the angiogenic properties of MCs depend on the angiogenic molecules contained in their secretory granules and indicate that FGF-2 and VEGF are the angiogenic cytokines primarily and perhaps synergistically, responsible for this vasoproliferative activity.

MAST CELLS IN HUMAN TUMOR ANGIOGENESIS In the uterine cervix, tryptase-positive MCs increase in number and vascularization increases in a linear fashion from dysplasia to invasive cancer.111 We have demonstrated that angiogenesis in human endometrial carcinoma is highly correlated with MC tryptase-positive cell counts and that these parameters increase with tumor progression.112 An association of VEGF and MCs with angiogenesis has been demonstrated in laryngeal carcinoma,113 in non small cell lung carcinoma where most intratumor MCs express VEGF,114-116 and in melanoma where MCs express both VEGF117 and FGF-2.118 Lastly, a

& $ % +( $%$+ % %$*

279

    #   $  @   !$   ‹$$ cell cancer of the oesophagus,119 squamous cell carcinoma of the oral cavity52 and in melanoma.51 Imada et al114 studied 85 cases of Stage I non small cell lung carcinoma and demonstrated a higher number of tryptase-positive MCs compared to cases of squamous cell carcinoma. A high correlation was observed between intratumor MC counts and microvessel counts; and double immuno-staining showed that most intratumor MCs expressed VEGF. In melanoma, MC accumulation around the margin of tumors has been observed to peak just as the tumor acquires the angiogenic phenotype and peri-tumor MC counts correlated strongly with microvascular density, melanoma progression and prognosis.51,117,118 Furthermore, in melanoma, MCs were closer to each other and to vessels.120 This close association between MCs and the endothelium may indicate that MCs are involved in the maintenance and long lasting functional integrity of the endothelium. Vascular density has been shown to be associated with a worse prognosis in Merkel cell carcinomas.121 Other factors associated with a worse outcome included tumor size, the    #!$ !  $ @ $ *#     of microvessel density and MC density correlated with the survival of Thai patients with primary colorectal cancer.122    !   % $ % microvessel density and MC density in the areas of highest vascular density. Patients %# $   %  !$   % @ $ #    $ !!  # # %# # !$  ## @ $   ! correlations between the microvessel density and MC counts in primary colorectal cancer were found also by Gulubova and Vlaykova.123 Patients with hypovascular tumor tissues #    $ !! # # %# # !$ $   $     %  !   ^! @   %# % @  #         # %# ## @ density. We have demonstrated that angiogenesis increases in parallel to the number of tryptase-positive MCs in lymph nodes from patients with breast cancer and that their !$     ##  #  %#      %# those without metastasis.124       #!  $ % the number of tryptase-positive MCs, the area occupied by MCs positive for tryptase, the microvascular density and the endothelial area in early breast cancer patients.125 Angiogenesis in benign lymphadenopathies and B-cell non-Hodgkin’s lymphomas (B-NHL), measured as microvessels counts, was correlated with the total and MC tryptase-positive counts. Both increased in step with the increase in malignancy grades.126,127 In NHL, the cellular expression of VEGF and FGF-2, as well as MC and vessel counts, has been assessed.58 The number of MCs were greater in T-cell lymphomas than in B-cell #–    ~`ƒ      % $ % ! $  number of MCs in healthy lung and between vessel count and number of VEGF-expressing  $ {$    ž=  ‚~  @   ! # MCs expressed VEGF mRN R A. These data suggest that MCs intervene in angiogenesis in these lesions by expressing VEGF. Glimelius et al128 evaluated the relationship between the number of MCs and the microvessel count in tissue samples from NHL-involved #   $##         %  ## microvessel count and the number of MCs. Angiogenesis represents an essential step of disease progression in other haematological malignancies. In Waldenström’s macroglobulinemia (WM), the bone marrow microvessel density is increased in 30-40% of patients, but this seems to have no impact on survival.129

280

MAST CELL BIOLOGY

Figure 2. Electron micrograph of a human MC from the lymph node in a patient with B-cell non-Hodgkin’s lymphoma. This MC is embedded in an area of intense angiogenesis and establishes a close contact with an endothelial cell precursor (ECP). Bar  1 +m.

Angiogenic cytokines produced by MCs, such as angiogenin, VEGF and FGF-2, are increased in the serum of WM or IgM- monoclonal gammopathies of undetermined   ˜ '™  †  %  !$   ! area and MC counts are highly correlated in patients with inactive and active multiple myeloma (MM) and in those with MGUS. Both parameters increase simultaneously in active MM.130 In both B-NHL and MM, MCs rest near or around blood or lymphatic capillaries (Fig. 2). Their ultrastructural picture includes piecemeal degranulation of their secretory granules, unlike the IgE-mediated massive degranulation that occurs during immediate hypersensitivity reactions.126,130-132 This morphology is typical of the slow degranulation that takes place in delayed hypersensitivity reactions and chronic

& $ % +( $%$+ % %$*

281

{133   { % $   !      %## favor chronic and progressive stimulation of MC degranulation.134 Bone marrow samples of patients with myelodysplastic syndromes display a high correlation between microvessel counts and both total and tryptase-positive MCs. In addition, both parameters increase simultaneously with tumor progression.135 There is also a correlation between the extent of angiogenesis and the number of tryptase-positive MCs in patients with early B-cell chronic lymphocytic leukaemia and tryptase-positive MCs predict the clinical outcome.136,137

DRUGS AFFECTING MAST CELL NUMBER Preliminary studies using anti-KIT antibodies,14 anti-TNF-_ antibodies6 or the MC stabilizer disodium cromoglycate (cromolyn)7 in mouse models, have demonstrated promising results even if administered after the initiation of tumor development. Treatment of mice bearing mammary adenocarinoma and pancreatic cancer with cromolyn led to clotting of blood vessels, hypoxia and tumor cell apoptosis.7,72 Unfortunately, cromolyn is a weak inhibitor of human MC secretion and is poorly adsorbed, so it is unlikely to be effective in treating cancer in humans. *#    < #  $  #  *” ˜ mesylate, Gleevec), has inhibitory activity against the signalling cascade activated through KIT (CD117).138 This inhibitory activity is the basis of the use of this drug against gastrointestinal stromal tumors (GIST), most of which harbour a KIT mutation.139 STI571 is also in use for some varieties of mastocytosis, although some KIT activating mutations involved in mastocytosis are resistant to its inhibitory activity.140 Ranitidine, an H2 receptor antagonist, when used as adjunctive therapy, prolonged survival of colorectal cancer patients.141 Another H2 receptor antagonist, famotidine, given pre-operatively, # $    #     #  ! changes in breast cancer in humans.142 Molica et al143 demonstrated that the reduction in the extent of bone marrow angiogenesis observed after sequential therapy with low doses  $$$ }$      {$  $ #  was associated to a reduction in the number of MCs . Dietary supplementation of silymarin, a naturally derived polyphenolic antioxidant, "       ~^ # ˜~=™^$ !   & albino male rats.143 NDEA administered rats showed increased MC density, along with upregulated expression of MMP-2 and MMP-9. Silymarin treatment inhibited the increase in MC density and downregulated the expression of MMP-2 and MMP-9 as revealed by &    $#  *#$    "     on liver carcinogenesis by attenuating the recruitment of MCs and thereby decreasing the expressions of MMP-2 and MMP-9.144

CONCLUSION MCs are found in multiple tissues and their presence in association with human cancer has been established for many years. However, multiple reports have described both a positive and a negative correlation between MC number and prognosis in various human tumor types (Fig. 3). These discrepancies are also observed in experimental studies, and

282

MAST CELL BIOLOGY

Figure 3. The dual role of MCs in tumor fate. MCs may release cytokines and growth factors in the tumor stroma, such as FGF-2, NGF, PDGF, IL-10 and IL-8, with detrimental effects to the host by stimulating tumor cell expansion. MCs are a major source of histamine, which can induce tumor cell proliferation through H1 receptors, while suppressing the immune system through H2 receptors. In addition, MCs synthesize and store angiogenic factors as well as matrix metalloproteinases, which promote tumor vascularization and tumor invasiveness, respectively. MCs may also generate immunosuppression by releasing IL-10, histamine and TNF-_. By contrast, MCs may promote inhibition of tumor cell  %# $    {   < $#  ƒ^ ƒ^’ ƒ^“  TNF-_. TNF-_, in particular, is effective in leukocyte chemoattraction. Chondroitin sulphate may # $  $–    $ # $   $  { # $# !   ^!   ˜\‚^  ^_™  !  ^_    %# factor-2; NGF, nerve growth factor; PDGF, platelet derived growth factor; TGF-`, transforming growth factor-`; TNF-_, tumor necrosis factor-_; INF-_, interferon-_; LTB4, leukotriene B4; MCP-3, MCP-4, mast cell protease-3, -4; MMP-2, MMP-9, matrix metalloproteinase-2, -9; VEGF, vascular endothelial growth factor; IL-1, IL-2, IL-6, IL-8, IL-10, interleukin-1, -2, -6, -8, -10.

could be due to differences in the mouse model used and the stage of carcinogenesis investigated. MCs are recruited in tumor development and play a key role in both angiogenesis and tissue remodelling by promoting both tumor initiation and growth. In addition, as tumor growth progresses, MCs recruit immune cells; or alternatively suppress antitumoral  ‚    @       $ $$  come from Huang and coworkers.13 *# } @     $     @ *$ ^ ! @  @     ! $   of the tumor microenvironment and tumor growth. MCs could be a new target for the adjuvant treatment of tumors, through the selective inhibition of angiogenesis, tissue remodelling and tumor-promoting molecules, permitting the secretion of cytotoxic cytokines and preventing MC-mediated immune suppression. Moreover, some of the new targeted anticancer therapies have pronounced effects on

& $ % +( $%$+ % %$*

283

MCs; in fact, it may be that some of their antitumor effects are closely related to their effect on MCs.

ACKNOWLEDGEMENTS Supported in part by MIUR R (PRIN 2007), Rome and Fondazione Cassa di Risparmio di Puglia, Bari, Italy.

REFERENCES 1. Ehrlich P. Beiträge zur Theorie und Praxis der histologischen Färbung. 1878, Thesis, Leipzig University. 2. Crivellato E, Beltrami C, Mallardi F et al. Paul Ehrlich’s doctoral thesis: a milestone in the study of mast cells. Br J Haematol 2003a; 123:19-21. 3. Galli SJ, Kalesnikoff J, Grimbaldeston MA et al. Mast cells as “tunable” effector and immunoregulatory cells: recent advances. Annu Rev Immunol 2005a; 23:749-86. 4. Galli SJ, Nakae S, Tsai M. Mast cells in the development of adaptive immune responses. Nat Immunol 2005b; 6:135-42.  @$ ƒ  ‚ && †     {    $^ $  $  squamous epithelial carcinogenesis. Genes Dev 1999; 13:1382-97. 6. Gounaris E, Erdman SE, Restaino C et al. Mast cells are an essential hematopoietic component for polyp development. Proc Natl Acad Sci USA 2007; 104:19977-82. 7. Soucek L, Lawlor ER, Soto D et al. Mast cells are required for angiogenesis and macroscopic expansion of Myc-induced pancreatic islet tumors. Nat Med 2007; 13:1211-8. 8. Nakayama T, Yao L, Tosato G. Mast cell-derived angiopoietin-1 plays a role in the growth of plasma cell tumors. J Clin Invest 2004; 114:1317-25. 9. Hart P, Townley S, Grimbaldeston M et al. Mast cells, neuropeptides, histamine and prostaglandins in UV-induced systemic immunosuppression. Methods 2002; 28:79-89. 10. Church M, Levi-Schaffer F. The human mast cell. J Allergy Clin Immunol 1997; 99:155-160. 11. Hart P, Grimbaldeston M, Finlay-Jones J. Immunosuppression and skin cancer: role of histamine and mast cells. Clin Exp Pharmacol Physiol 2001; 28:1-8. 12. Okayama Y, Kawakami T. Development, migration and survival of mast cells. Immunol Res 2006; 34:97-115. Œ }    ~<       #      #  { Immunol Rev 2007; 217:304-28. ’ `$ † ƒ    #   @^       ! "  # {  $$   $  !  † _••—– __“€^”€  ~%< =@ &! @* *$  `   ƒ^€     *#”^ ! {   > =" Med 2009; 206:1653-60. 16. Sebolt-Leopold JS, Herrera R. Targeting the mitogen-activated protein kinase cascade to treat cancer. Nat Rev Cancer 2004; 4:937-47. ” % #    & ~^<† <   {^   @ † *# 2004; 3:1214-6. 18. Pardoll D. Does the immune system see tumors as foreign or self? Annu Rev Immunol 2003; 21:807-39. 19. Munn DH, Mellor AL. The tumor-draining lymph node as an immune-privileged site. Immunol Rev 2006; 213:146-58. 20. Ullrich SE, Nghiem DX, K Khaskina P. Suppression of an established immune response by UVA—a critical role for mast cells. Photochem Photobiol 2007; 83:1095-100. 21. Grimbaldenston MA, Nakae S, Kalesnikoff K et al. Mast cell-derived interleukin 10 limits skin pathology in contact dermatitis and chronic irradiation with ultraviolet B. Nat Immunol 2007; 38:1095-104. 22. Lu LF, Lind EF, Gondek DC et al. Mast cells are essential intermediaries in regulatory T-cell tolerance. Nature 2006; 442:997-1002. 23. K Kripke M. Immunological unresponsiveness induced by ultraviolet radiation. Immunol Rev 1984; 80:87-102. 24. Ch’ng S, Wallis RA, Yuan L. Mast cells and cutaneous malignancies. Modern Pathol 2006; 19:149-59.

284

MAST CELL BIOLOGY

25. Wille J, Kydonieus K A, Murphy G. Cis-urocanic acid induces mast cell degranulation and release of preformed TNF-alpha: a possible mechanism linking UVB and cis-urocanic acid to immunosuppression of contact hypersensitivity. Skin Pharmacol Physiol 1999; 12:18-27. 26. Chen W, Jin W, Hardegen N et al. Conversion of peripheral CD4 CD25< naïve T-cells to CD4 CD25

regulatory T-cells by TGF-beta induction of transcription factor Foxp3. J Exp Med 2003; 198:1875-86. 27. Kambayashi T, Allenspach EJ, Chang JT et al. Inducible MHC class II expression by mast cells supports effector and regulatory T-cell activation. J Immunol 2009; 182:4686-95. 28. Ju MJ, Qiu SJ, Gao Q et al. Combination of peritumoral mast cells and T-regulatory cells predicts prognosis of hepatocellular carcinoma. Cancer Sci 2009; 100:1267-74. 29. Conti P, Pang X, Boucher W et al. Impact of Rantes and MCP-1 chemokines on in vivo basohilic mast cell recruitment in rat skin injection model and their role in modifying the protein and mRN R A levels for histidine decarboxylase. Blood 1997; 89:4120-7. 30. Theoharides T, Conti P. Mast cells: the Jekyll and Hyde of tumor growth. Trends Immunol 2004; 25:235-41. 31. Dabbous M, Walker R, Haney L et al. Mast cells and matrix degradation at sites of tumor invasion in rat mammary adenocarcinoma. Br J Cancer 1986; 54:459-65. 32. Stack MS, Johnson DA. Human mast cell tryptase activates single-chain urinary-type plasminogen activator (pro-urokinase). J Biol Chem 1994; 269:9416-9. 33. Baram D, Vaday G, Salamon P et al. Human mast cells release metalloproteinase-9 on contact with activated T-cells: juxtacrine regulation by TNF-alpha. J Immunol 2001; 167:4009-16. 34. Hara M, Matsumori A, Ono K et al. Mast cells cause apotosis of cardiomyocytes and proliferation of other intramyocardial cells in vitro. Circulation 1999; 100:1443-9. 35. He S, a Walls AF. Human mast cell chymase induces the accumulation of neutrophils, eosinophils and # {    !! † > \#  €——– _’€^•• 36. Sakamoto S, Goldhaber P, Glimcher M. The effect of heparin on the amount of enzyme released in tissue culture and on the activity of the enzyme. Calcif Tissue Int 1973; 12:247-58. 37. Markwardt F, Klocking H. Heparin-induced release of plasminogen activator. Haemostasis 1977; 6:370-4. 38. Niwa Y, Kasugai T, Ohno K et al. Anemia and mast cells depletion in mutant rats that are homozygous at ‘white spotting (Ws)’ locus. Blood 1991; 78:1936-41. 39. Lazar-Molnar E, Hegyesi H, Pallinger E et al. Inhibition of human primary melanoma cell proliferation by histamine is enhanced by interleukin-6. Eur J Clin Invest 2002; 32:743-9. 40. Burtin C, Ponvert C, Fray A. Inverse correlation between tumor incidence and tissue histamine levels in W/Wv, W/ and / mice. J Natl Cancer Inst 1985; 74:671-4. 41. Halaban R, K Kwon BS, Ghosh S et al. bFGF as an autocrine growth fator for human melanomas. Oncogene Res 1988; 3:177-86. 42. Schadendorf D, Moller A, Algermissen B et al. IL-8 produced by human malignant melanoma cells in vitro is an essential autocrine growth factor. J Immunol 1993; 151:2667. 43. Mukheriee S, Bandyopadhyay G, Dutta C et al. Evaluation of endoscopic biopsy in gastric lesions with a     #        > \#   _••€– __•^’ 44. Imai T, Michizawa M, Shimizu H et al. Bilateral multiple spindle cell lipomas of the tongue. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 106:264-9. ’ ~<#  š ~ š# š       $     # <  >  # 2008; 30:278-280. ’“ ƒ  @$·$   @$·$              ‚ >  # Embryol 2009; 50:85-90. 47. Aoki M, Pawankar R, Niimi Y et al. Mast cells in basal cell carcinoma express VEGF, IL-8 and RAN A TES. Int Arch Allergy Immunol 2003; 130:216-23. 48. Oliveira CR, Albuquerque GC, Simon EF et al. Case for diagnosis. Telangectasia macularis eruptive perstans. An Bras Dermatol 2009; 84:87-9. 49. Kiliç A, Kösem M, Demirok A et al. Conjunctival myxoma: a clinicopathologic report. Ophthalmic Surg Lasers Imaging 2008; 39:514-6. •  ! ‚ $ ' $ ‚   !     ^     # and molecular analysis. Cancer Cytopathol 2009; 117:128-36. 51. Ribatti D, Ennas MG, Vacca A et al. Tumor vascularity and tryptase positive-mast cells correlate with a poor prognosis in melanoma. Eur J Clin Invest 2003b; 33:420-5. 52. Iamaroon A, Pongsirlwet S, Jittidecharaks S et al. Increase of mast cells and tumor angiogenesis in oral squamous cell carcinoma. J Oral Pathol Med 2003; 32:195-9. 53. Rojas IG, Spencer ML, Martinez A et al. Characterization of mast cell subpopulations in lip cancer. J Oral Pathol Med 2005; 34:268-73.

& $ % +( $%$+ % %$*

285

54. Sharma VK, Agrawal AK, Pratarp VK K et al. Mast cell reactivity in lymphoma: a preliminary communication. India J Cancer 1992; 29:61-5.    =   $           %#     `<§ lymphoma. Br J Haematol 2002; 119:122-4. 56. Molin D. Bystander cells and prognosis in Hodgkin lymphoma. Review based on a doctoral thesis. Ups J Med Sci 2004; 109:179-228. ” $<$# ` ˆ#%  <$  š      $   †^ #– #     Histopathology 2006; 49:498-505. 58. Fukushima N, Satoh T, Sano M et al. Angiogenesis and mast cells in nonHodgkin’s lymphoma: strong correlation in angioimmunoblastic T-cell lymphoma. Leuk Lymphoma 2001; 42:709-20. 59. Dave SS, Wright G, Than B et al. Prediction of survival in follicular lymphoma based on molecular features  $ ^   $  ~ = >  _••’– Œ_€^“€ 60. Nechushtan H, Razin E. The function of MIFT and associated proteins in mast cells. Mol Immunol 2002; 38:1177-80. 61. Tournilhac O, Santos DD, Xu L et al. Mast cells in Waldenstrom’s macroglobulinemia support lymphoplasmacytic cell growth through CD154/CD40 signaling. Ann Oncol 2006; 17:1275-82. “_ ~$  ~ *< ` ~#$       $         biopsy specimens leads to a better prognosis of prostate cancer. Br J Cancer 2007; 97:952-6. 63. Fleiscmann A, Schlomm T, Köllermann J et al. Immunological microenvironment in prostate cancer: high mast cell densities are associated with favorable tumor characteristics and good prognosis. Prostate 2009; 69:976-81. 64. Gomes AP, Johann JE, Lovato GG et al. Comparative analysis of the mast cell density in normal oral mucosa, actinic cheilitis and lip squamous cell carcinoma. Braz Dent J 2008; 19:186-9. 65. Lago Costa N, Ferreira Oton-Lelte A, Pelxodo Chelm-Júnlor A et al. Density and migration of mast cells in lip squamous cell carcinoma and actinic cheilitis. Histol Histopathol 2009; 24:457-65. 66. Rojas IG, Martinez A, Brethauer U et al. Actinic cheilitis: epithelial expression of COX-2 and its association with mast cell tryptase and PAR-2. Oral Oncol 2009; 45:284-90. 67. Oliveira-Neto HH, Ferreira Leite A, Lago Costa N et al. Decrease in mast cells in oral squamous cell carcinoma: Possible failure in the migration of these cells. Oral Oncol 2007; 43:484-90. 68. Aaltomaa S, Lipponen P, Papinaho S et al. Mast cells in breast cancer. Anticancer Res 1993; 13:785-8. “€    `$  < ! ~   *#           $  !! breast cancers with a favorable prognosis. Mod Pathol 2004; 17:690-5. 70. Gooch SJ, Lee AV, Yee D. Interleukin 4 inhibits growth and induces apoptosis in human breast cancer cells. Cancer Res 1998; 58:4199-205. 71. Ohno S, Inagawa H, Soma G et al. Role of tumor-associated macrophage in malignant tumors: should the   #     #  <  $ $  !$›  ‚ _••_– 22:4269-75. 72. Samoszuk M, Corwin MA. Mast cell inhibitor cromolyn increases blood clotting and hypoxia in murine breast cancer. Int J Cancer 2003; 107:159-63. 73. Kankkunen JP, Harvima IT, Naukkarinen A. Qunatitative analysis of tryptase and chymase containing mast cells in benign and malignant breast lesions. Int J Cancer 1997; 72:385-8. 74. Thorense S, Tangen M, Hartveit F. Mast cells in the axillary nodes of breast cancer patients. Diagn Histopathol 1982; 5:65-7. 75. Della Rovere F, Granata A, Monaco M et al. Phagocytosis of cancer cells by mast cells in breasr cancer. Anticancer Res 2009; 29:3157-61. 76. Amini RM, Aaltonen K, Nevanlinna H et al. Mast cells and eosinophils in invasive breast carcinoma. BMC Cancer 2007; 7:165-9. 77. Welsh TJ, Green RH, Richardson D et al. Macrophage and mast cell invasion of tumor cell islets confers a marked survival advantage in non small-cell lung cancer. J Clin Oncol 2005; 23:8959-67. 78. Chan JK, Magistris A, Loizzi V et al. Mast cell density, angiogenesis, blood clotting and prognosis in women with advanced ovarian cancer. Gynecol Oncol 2005; 99:20-5. 79. Cinel L, Aban M, Bastruk M et al. The association of mast cell density with myometrial invasion in endometrial carcinoma: a preliminary report. Pathol Res Pract 2009; 205:255-8. 80. Alì G, Boldrini L, Lucchi M et al. Tryptase mast cells in malignant pleural mesothelioma as an independent favorable prognostic factor. J Thorac Oncol 2009; 4:348-54. 81. Thorton SC, Mueller SM, Levine EM. Human endothelial cells: use of heparin in cloning and long term cultivation. Science 1983; 222:623-5. 82. Alessandri G, Raju KS, Gullino PM. Characterization of a chemoattractant for endothelium induced by angiogenic effectors. Cancer Res 1984; 44:1579-84. 83. Ribatti D, Roncali L, Nico B et al. Effects of exogenous heparin on the vasculogenesis of the chorioallantoic membrane. Acta Anat 1987; 130:257-63.

286

MAST CELL BIOLOGY

84. Norrby K, Sorbo J. Heparin enhances angiogenesis by a systemic mode of action. Int J Exp Pathol 1992; 73:1451-5. 85. Norrby K. Heparin and angiogenesis: a low molecular weight fraction inhibits and a high molecular weight fraction stimulates angiogenesis systematically. Haemostasis 1993; 23:144-9. 86. Jakobson AM, Hahnenberger R. Antiangiogenic effect of heparin and other sulphated glycosaminoglycans in the chick embryo chorioallantoic membrane. Pharmacol Toxicol 1991; 69:122-6. 87. Wilks JW, Scott PS, Urla LK K et al. Inhibition of angiogenesis with combination treatments of angiostatic steroids and suramin. Int J Radiat Biol 1991; 60:73-7. 88. Castellot JJ, Karnovsky MJ, Spiegelman BM. Differentiation-dependent stimulation of neovascularization and endothelial cell chemotaxis by 3T3 adipocytes. Proc Natl Acad Sci USA 1982; 79:5597-5601. 89. Taylor S, Folkman J. Protamine is an inhibitor of angiogenesis. Nature 1982; 297:307-12. 90. Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992; 267:10931-4. 91. Yayou A, Klagsbrun M, Esko JD et al. Cell surface, heparin-like molecules are required for binding of     %#    ##     @ €€– “’—’^— 92. Sörbo J, Jakobsson A, Norrby K. Mast-cell histamine is angiogenic through receptors for histamine1 and histamine2. Int J Exp Pathol 1994; 75:43-50. 93. Thompson WD, Campbell R, Evans T. Fibrin degradation and angiogenesis: quantitative analysis of the angiogenic response in the chick chorioallantoic membrane. J Pathol 1995; 145:27-37. 94. Fang KC, Wolters PJ, Steinhoff M et al. Mast cell expression of gelatinase A and B is regulated by kit ligand and TGF-`. J Immunol 1999; 162:5528-35. €    ~ &<    !    !! "     

\^  #$ mast cells. Dev Immunol 2000; 7:131-42. 96. Vincent AJ, Zhang J, Ostor A et al. Matrix metalloproteinase-1 and-3 and mast cells are present in the endometrium of women using progestin-only contraceptives. Human Reprod 2000; 15:123-30. 97. Raza SL, Cornelius LA. Matrix metalloproteinases: pro- and anti-angiogenic activities. J Invest Dermatol Symp Proc 2000; 5:47-54. 98. Taiplae J, Lohi J, Saarinen J et al. Human mast cell chymase and leukocyte elestase release latent transforming growth factor beta-1 from the extracellular matrix of cultured human epithelial and endothelial cells. J Biol Chem 1995; 270:4689-96. 99. Gruber BL, Marchese MJ, Suzuki K et al. Synovial procollagenase activation by human mast cell tryptase dependence upon matrix metalloproteinase 3 activation. J Clin Invest 1989; 84:1657-62. 100. Blair RJ, Meng H, Marchese MJ et al. Tryptase is a novel, potent angiogenic factor. J Clin Invest 1997; 99:2691-2700. 101. Muramatsu M, Katada J, Hattori M et al. Chymase mediates mast cell-induced angiogenesis in the hamster sponge granuloma. Eur J Pharmacol 2000a; 402:181-91. 102. Muramatsu M, Katada J, Hattori M et al. Chymase as a proangiogenic factor; a possible involvement of chymase-angiotensin-dependent pathway in the hamster sponge angiogenesis model. J Biol Chem 2000b; 275:5545-52. •Œ Ÿ$    ‚> # \   '  $$  $}      %#  in mast cell secretory granules: morphological evidence for bFGF release through degranulation. J Histochem Cytochem 1998; 46:1119-28. 104. Grutzkau A, Kruger K -Krasagakes K S, Baumeister H et al. Synthesis, storage and release of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) by human mast cells: implications  #     ž=  _•“  † @ €€—– €—”^—’ 105. Cantarella G, Lempereur L, Presta M et al. Nerve growth factor-endothelial cell interactions lead to angiogenesis in vitro and in vivo. FASEB J 2002; 16:1307-1309. 106. Emanueli C, Salis MB, Pinna A et al. Nerve growth factor promotes angiogenesis and arteriogenesis in ischemic hindlimbs. Circulation 2002; 106:2257-62. 107. Kessler DA, Langer RS, Pless NA et al. Mast cells and tumor angiogenesis. Int J Cancer 1976; 18:703-9. 108. Poole TJ, Zetter BR. Stimulation of rat peritoneal mast cell migration by tumor-derived peptides. Cancer Res 1983; 43:5857-61. •€  < >‚ @ % \ *$    ^  &&v mice exhibit a decreased rate of tumor angiogenesis. Int J Cancer 1988; 42:48-52. 110. Ribatti D, Crivellato E, Candussio L et al. Mast cells and their secretory granules are angiogenic in the chick embryo chorioallantoic membrane. Clin Exp Allergy 2001; 31:602-8. 111. Benitez-Bribiesca L, Wong A, Utrera D et al. The role of mast cell tryptase in neoangiogenesis of premalignant and malignant lesions of the uterine cervix. J Histochem Cytochem 2001; 49:1061-2. 112. Ribatti D, Finato N, Crivellato E et al. Neovascularization and mast cells with tryptase activity increase simultaneously with pathologic progression in human endometrial cancer. Am J Obstet Gynecol 2005; 193:1961-5.

& $ % +( $%$+ % %$*

287

113. Sawatsubashi M, Yamada T, Fukushima N et al. Association of vascular endothelial growth factor and mast cells with angiogenesis in laryngeal squamous cell carcinoma. Virchows Arch 2000; 436:243-8. 114. Imada D, Shijubo N, Kojima H et al. Mast cells correlate with angiogenesis and poor outcome in stage I lung adenocarcinoma. Eur Resp J 2000; 15:1087-93. 115. Takanami I, Takeuchi K, Narume M. Mast cell density is associated with angiogenesis and poor prognosis in pulmonary adenocarcinoma. Cancer 2000; 88:2686-92. 116. Tomita M, Matsuzaki Y, Onitsuka T. Effect of mast cell on tumor angiogenesis in lung cancer. Ann Thorac Surg 2000; 69:1686-90. 117. Toth T,Toth-Jakatics R, Jimi S et al. Cutaneous malignant melanoma: correlation between neovascularization and peritumor accumulation of mast cells overexpressing vascular endothelial growth factor. Hum Pathol 2000; 31:955-60. — ‚  ž  ‚ ‚   ~!$ } "      %#  ^_   cell with tryptase activity increase simultaneously with pathological progression in human malignant melanoma. Eur J Cancer 2003; 39:666-75. 119. Elpek GO, Gelen T, Aksoy NH et al. The prognostic relevance of angiogenesis and mast cells in squamous cell carcinoma of the oesophagus. J Clin Pathol 2001; 54:940-4. 120. Guidolin D, Crivellato E, Nico B et al. An image analysis of the spatial distribution of perivascular mast cells in human melanoma. Int J Mol Med 2006; 17:981-7. 121. Ng L, Beer TW, Murray K. Vascular density has prognostic value in Merkel cell carcinoma. Am J Dermatopathol 2008; 30:442-5. __ š!$#  *‘  \$^   \      !    cell density for the survival of Thai patients with primary colorectal cancer. J Med Assoc Thai 2008; 91:723-32. _Œ $$!  ž<! * \       $   !$   # survival of patients with primary colorectal cancer. J Gastroenterol Hepatol 2009; 24:1265-75. 124. Ribatti D, Finato N, Crivellato E et al. Angiogenesis and mast cells in human breast cancer sentinel lymph nodes with and without micrometastases. Histopathology 2007; 51:837-42. 125. Ranieri G, Ammendola M, Patruno R et al. Tryptase-positive mast cells correlate with angiogenesis in early breast cancer patients. Int J Oncol 2009; 35:115-20. 126. Ribatti D, Nico B, Vacca A et al. Do mast cells help to induce angiogenesis in B-cell nonHodgkin’s lymphomas? Br J Cancer 1998; 77:1900-6. 127. Ribatti D, Vacca A, Marzullo A et al. Angiogenesis and mast cell density with tryptase activity increase simultaneously with pathological progression in B-cell nonHodgkin’s lymphomas. Int J Cancer 2000; 82:171-5. 128. Glimelius I, Edstrom A, Fischer M et al. Angiogenesis and mast cells in Hodgkin’s lymphoma. Leukemia 2005; 19:2360-2. 129. Terpos E, Tasidou A, Kastritis E et al. Angiogenesis in Waldenström’s macroglobulinemia. Clin Lymphoma Myeloma 2009; 9:46-9. 130. Ribatti D, Vacca A, Nico B et al. Bone marrow angiogenesis and mast cell density increase simultaneously with progression of human multiple myeloma. Br J Cancer 1999; 79:451-5. 131. Crivellato E, Nico B, Vacca A et al. Mast cell heterogeneity in B-cell nonHodgkin’s lymphomas: an ultrastructural study. Leuk Lymphoma 2002; 43:2201-5. 132. Crivellato E, Nico B, Vacca A et al. Ultrastructural analysis of mast cell recovery after secretion by piecemeal degranulation in B-cell nonHodgkin’s lymphoma. Leuk Lymphoma 2003; 44:517-21. 133. Kops SK, Van Loveren H, Rosenstein RW et al. Mast cell activation and vascular alterations in immediate hypersensitivity-like reactions induced by a T-cell derived antigen-binding factor. Lab Invest 1984; 50:421-34. 134. Theoharides T, Kempuraj D, Tagen M et al. Differential release of mast cells mediators and the pathogenesis  { $ ‚! _••”– _”“^”— 135. Ribatti D, Polimeno G, Vacca A et al. Correlation of bone marrow angiogenesis and mast cells with tryptase activity in myelodysplastic syndromes. Leukemia 2002; 16:1680-4. 136. Molica S, Vacca A, Crivellato E et al. Tryptase-positive mast cells predict clinical outcome of patients with early B-cell chronic lymphocytic leukemia. Eur J Haematol 2003; 71:137-9. 137. Ribatti D, Molica S, Vacca A et al. Tryptase-positive mast cells correlate positively with bone marrow angiogenesis in B-cell chronic lymphocytic leukemia. Leukemia 2003; 17:1428-30. Œ— ` # @  # >  $< †>   #  ^<    < !  * ” a selective tyrosine kinase inhibitor. Blood 2000; 96:925-32. 139. Kitamura Y, Hirotab S. Kit as a human oncogenic tyrosine kinase. Cell Mol Life Sci 2004; 61:2924-31. 140. Akin C, Metcalfe DD. The biology of Kit in disease and the application of pharmacogenetics. J Allergy Cin Immunol 2004; 114:13-9.

288

MAST CELL BIOLOGY

141. Nielsen HJ, Christenseb IJ, Svendsen MN et al. Ranitidine as adjuvant treatment in colorectal cancer. Br J Surg 2002; 89:1416-22. ’_ \ # ‚   $    # $    #     › Results of a randomized prospective pilot study. Acta Oncol 2002; 41:362-5. 143. Molica S, Montillo M, Ribatti D et al. Intense reversal of bone marrow angiogenesis after sequential {$ ^$  $}$^ #   ! #  # $< Haematologica 2007; 92:1367-74. 144. Ramakrishnan G, Jagan S, Kamaraj S et al. Silymarin attenuated mast cell recruitment thereby decreased the expressions of matrix metalloproteinases-2 and 9 in rat liver carcinogenesis. Invest New Drugs 2009; 27:233-40.

INDEX

A a1 37, 51, 53, 54, 56 a4b7 3, 14, 17, 18, 21 a4 integrin 18, 19 ABT-737 56 Activation 2-9, 23-25, 29-42, 47, 49, 50, 52-55, 62-63, 66-72, 74, 75, 77-80, 91, 93, 94, 100, 103, 107-110, 113, 116, 124, 126, 127, 130-133, 135, 137, 138, 143, 144, 146-156, 165, 166, 169, 172, 174, 187, 191-199, 201, 204, 215, 216, 218, 219, 222, 224-227, 236-240, 244-249, 252, 254-260, 271-274, 276, 282 Adaptive immunity 107, 162, 163, 174-176 Aggregation 5-7, 30-32, 34-39, 42, 54, 64, 66, 91, 93, 97, 100, 103, 108, 116, 127, 144, 148-150, 153, 154, 191, 192 Airway smooth muscle (ASM) 48, 188, 214, 225, 237, 238, 242-244, 249-252, 254, 255, 257, 258   { Œ  ’ ŒŒ 150, 201, 212, 218, 222-224, 226, 252 Allergy 29, 42, 49, 55, 56, 107, 132, 143, 146, 155, 162, 163, 176, 212, 248 __¸   “ “—

Angiogenesis 5, 48, 124, 133, 154, 218, 226, 238, 240, 260, 270-272, 277-282 Angiotensin 212, 219-221, 226, 227, 237, 251, 277 Antigen 5, 6, 7, 14, 15, 21-24, 29-35, 40, 41, 47, 62-64, 66, 67, 69, 70, 73, 74, 76-79, 91-94, 97-99, 103, 113, 123, 127, 128, 132, 133, 144, 146-148, 152, 154, 167, 172, 174, 175, 186-188, 191-198, 201-204, 239, 251, 252, 255-257, 274 Apoptosis 3, 4, 6, 20, 31, 33, 34, 36, 37, 39, 40, 47, 49-53, 55, 56, 76, 125, 146, 150, 188, 195, 226, 273, 275, 276, 281, 282 Apoptotic protease activating factor 1 (APAFP 1) 50, 53

B Bacteria 29, 166, 169, 173, 198, 224, 245, 246 Bad 51, 52, 54, 56, 220 Basic FGF (bFGF) 240, 244, 248, 258, 259 Basophil 15, 16, 33, 34, 63, 65, 91, 94, 99, 145, 151, 153, 154, 156, 191, 214, 237-239, 256 Bax 4, 50-53 Bcl-2 3, 4, 36, 47, 50-56 Bcl-xL 36, 37, 51, 53-56 289

290

†{^  ’ “ Bim 4, 36, 37, 51-55 Bronchial hyperresponsiveness (BHR) 134, 214, 237, 248-252, 254 Bronchoalveolar lavage (BAL) 237, 246, 248, 249, 258, 259

C Ca2+ 8, 31, 32, 38, 62-81, 99, 148, 151, 155, 247 Calcitonin gene-related peptide (CGRP) 165, 213 Calcium channel 8, 130, 135 Calcium mobilization 100, 130, 131 Carboxypeptidase A3 (CPA P 3) 190, 214, 215 Caspase 3, 37, 49-51, 53, 54, 146 Cathepsin C 227 Cathepsin G 214, 217, 220, 221, 223, 226, 227, 241 CCL2 3, 5, 14, 22, 23, 32, 40, 128, 155, 169, 193, 238, 247, 271 CCR2 3, 14, 18, 22, 23 CD11B 20 CD48 164, 168, 245 CD200 Receptor (CD200R) 145, 153, 156 CD300a 145, 150, 151, 156 Cell adhesion 18, 37, 147, 154, 237, 251, 252, 259 Cellular adhesion molecule 18, 25 Chemokine 5, 25, 30-34, 91, 107, 144, 165-169, 187-189, 192, 199, 236, 238, 241, 242, 251, 256, 258, 260, 271 Chemokine receptor 3, 14, 17, 18, 22, 23, 25, 33, 237, 240 Chemotaxin 6, 22, 32, 63, 74, 76, 77, 124, 132, 133, 143, 146, 155, 193, 237-239 Chymase 4, 5, 49, 136, 169, 201, 212, 214-223, 226, 227, 237, 240, 248, 250, 251, 254, 271, 276-278 Co-aggregation 144, 148, 150, 153, 154

MAST CELL BIOLOGY

Collectin 165, 167, 168 Complement 67, 144, 146, 164, 165, 187, 191, 193, 196, 197, 254 Cross-linking 5, 30, 35, 144, 151, 212, 254 C-type lectin superfamily 145, 147, 154 CXCR2 3, 14, 18, 22, 24 Cytokine 3-6, 8, 15, 23, 24, 30-34, 36-38, 40, 41, 48-50, 52-54, 62, 63, 70, 74, 91, 102, 107, 110, 123, 124, 127, 128, 130, 134, 138, 144, 146, 148-151, 153-155, 164-171, 187-189, 191-199, 201, 202, 204, 212, 217, 223, 235-237, 239-242, 247-249, 252, 254, 256-259, 273, 274, 276, 278, 280, 282

D Degranulation 4, 6, 8, 30-34, 37, 38, 54, 62, 63, 65-67, 69, 70, 72-78, 80, 91, 97-100, 102, 103, 108-116, 123, 124, 127-135, 137, 148-151, 153-155, 164, 170, 192-196, 203, 204, 212-214, 217, 222, 225, 242, 246-249, 252, 255, 258, 260, 271, 272, 274, 280, 281 Dendritic cell 3, 14, 17, 19, 20, 24, 125, 133, 150-152, 163-165, 174, 186, 193, 237, 238, 240, 245, 256 Dephosphorylation 116, 124, 147, 151, 155 Differentiation 15, 16, 29, 33, 40-42, 123, 126, 133, 136, 146, 151, 152, 188, 193, 195, 196, 203, 238-240, 252, 256, 257 Dipeptidyl peptidase I (DPPI) 214, 227 Disease 2-4, 9, 29, 30, 32, 35, 38, 41, 42, 47-49, 52, 55, 56, 62, 65, 69, 77-80, 107, 118, 119, 123, 124, 126, 133, 134, 136, 138, 139, 143, 146, 155, 156, 162, 165, 171-173, 176, 186, 192, 199, 201, 202, 204, 205, 222, 225, 235, 240, 241, 246-248, 250, 252, 254, 256-260, 270, 279

INDEX

E Early asthmatic response (EAR) 248, 249, 255 Eicosanoid 5, 30, 62, 63, 67, 76, 127, 168 Endothelin 168, 171, 173, 197, 198, 212, 215-218 Eosinophilic bronchitis (EB) 249, 250 Epidermis 174, 193, 200, 274 Exocytosis 4, 107, 108, 110-118, 131, 169, 170, 227 Extracellular matrix (ECM) 15, 153, 214, 226, 237, 239, 251, 259, 274, 275, 277 Extracellular signal-related kinase (ERK) 33, 35-38, 77, 144, 148, 149, 154, 155

F Fas 4, 37, 49, 50, 53, 146 ¹   ’ ’” ’€ • _ 156, 192, 202 FGF-2 240, 277-280, 282 FOXO3a 4, 52 Fyn 7, 31, 37, 42, 66-68, 115, 127, 130, 132, 155

G G-protein coupled receptors (GPCRs) 6-8, 32, 94, 124, 132, 166 Granule 4, 5, 14, 15, 34, 49, 65, 107-110, 112, 113, 115, 117, 118, 131, 134, 136, 168-170, 190, 192-194, 198, 212, 219, 226, 227, 246, 258, 259, 277, 278, 280

H Hck 31, 37, 42 Heterogeneity in IgE 34, 35, 40, 41, 92, 109, 128, 237

291

`#     = ˜‚™ 5-8, 15, 17, 20, 29-42, 47, 54, 55, 64, 66, 74, 91-103, 107-110, 116, 118, 126-128, 130-135, 138, 143, 144, 146, 148-156, 170, 174, 188, 191, 192-195, 199, 201, 204, 212, 239, 245, 247, 252, 254 Histamine 4, 30, 31, 34, 65, 76, 91, 107, 110, 111, 114, 128, 134-138, 146, 150, 163, 165, 168-170, 188, 192-194, 196, 199, 204, 213, 214, 224-226, 236, 237, 239, 247-249, 251, 252, 254-260, 272-277, 282 Human lung mast cell (HLMC) 4, 33, 36, 217, 235, 236, 252, 255, 259 Hypotension 123, 137, 215

I # $    _— Ig-like super-family 147 Immunoglobulin E (IgE) 5, 6, 15, 23-25, 29-42, 47, 54, 55, 63, 70, 74, 91, 94-99, 101, 102, 107-109, 114, 116, 126, 127, 134, 135, 137, 143, 144, 146, 148-152, 155, 156, 162, 170, 172, 186-188, 191-193, 195, 196, 198-201, 204, 212, 217, 222, 237, 239, 240, 246-249, 251, 252, 254-257, 280 IgE receptor 15, 29, 91, 94, 95, 99, 102, 107, 144, 170, 212, 222, 240 IgE Fc receptor 191 Immunoreceptor tyrosine-based activation motif (ITAM) 7, 31, 37, 91, 143, 144, 146, 148, 149, 151 ITAMi 147, 149 Immunoreceptor tyrosine-based inhibition motif (ITIM) 8, 143, 144-155 Infection 14, 20, 24, 25, 29, 32, 39, 40, 42, 48, 49, 162-165, 169-172, 174-176, 196-199, 212, 218, 223, 224, 227, 235, 245-247, 257, 270

292

{ Œ  ’ _•^_ Œ_ ’”^’€ 55, 56, 123, 125, 132-134, 138, 146, 150, 152, 165, 169-174, 187, 192, 198, 199, 201, 202, 204, 212, 213, 217-219, 222-228, 235, 245, 247-249, 251, 252, 254-259, 271, 272, 275, 281, 282 Inhibitory receptor 94, 143, 145-153, 155, 156 Innate immunity 6, 29, 42, 162, 165, 170, 212 Integrin 3, 14, 15, 17-21, 25, 144, 151, 152, 165, 168 Interleukin-9 273

K KIT 3, 5-9, 15-17, 20, 40, 52, 55, 63, 73, 146, 151, 155, 170, 189-191, 200, 203, 218, 252, 272, 274, 276, 281

L Late asthmatic response (LAR) 248, 249 Leukocyte-associated Ig-like receptors-1 (LAIR-1) 145, 147, 152, 153 Leukocyte immunoglobulin receptor (LIR) 147 Leukotriene (LT) 5, 30, 31, 34, 91, 130, 146, 165, 169, 188, 192, 236, 240, 241, 245, 246, 282 Linker for activation of T cells (LAT) 7, 37, 38, 66, 100, 103, 148, 149, 151 L Lymphocyte 16, 17, 19, 20, 53, 74, 78, 79, 124-126, 152, 154, 174, 175, 194-198, 219, 227, 238, 246, 257, 281 L Lyn 7, 31, 37, 38, 42, 66-68, 94, 102, 103, 127, 144, 148

MAST CELL BIOLOGY

Mastin 214 Mastocytosis 9, 40, 47, 55, 222, 281 Matrix metalloproteinase (MMP) 212, 214, 218, 219, 226, 271, 272, 275, 277, 281, 282 Mcl-1 51, 53-56 Membrane 4-8, 31, 34, 35, 38, 50, 51, 62-76, 78-80, 91-103, 107-109, 111, 113-115, 117, 118, 123-125, 131, 135, 144, 153, 154, 166, 169-171, 194, 196, 212, 227, 237, 249, 252, 258, 259, 271, 277, 278 Membrane fusion 4, 34, 107-109, 111, 117, 118 Microdomain 7, 34, 35, 80, 92, 95-97 Mitochondria 51, 62-65, 75, 76, 78, 146 Monoclonal vs Polyclonal IgE 41 Monomeric IgE 6, 29-37, 39-42, 254 Mucosal addressin cellular adhesion molecule (MAdCAM)-1 3, 14, 18, 21 Myeloid lineage 30

N Natural killer T cell (NKT) 19, 23, 24, 187, 236 Neurolysin 214-216 Neurotensin 146, 165, 166, 168, 171, 173, 198, 212, 215, 216 Neutrophil 15, 22, 31-33, 48, 134, 145-147, 152, 154, 162, 164, 165, 171, 189, 197, 198, 201, 202, 217, 218, 221, 223, 224, 226, 227, 237-239, 245, 246, 272 ~^º† “ _’“ _’” _ Non-T-cell activation linker (NTAL) 7, 38 Notch 195

M

O

Mast cell (MC) 2-9, 14-25, 29-42, 47-49, 52-56, 62-80, 91, 92, 94, 99, 102, 103, 107-119, 123-138, 143-156, 162-166, 168-176, 186-205, 212-228, 235-260, 270-283 Mast cell function-associated antigen (MAFA) 94, 145, 154, 155

Orai 8, 64-66, 68, 71-73, 78-81

P Paired Ig-Llike receptor B (PIR-B) 145, 152

INDEX

Passive cutaneous anaphylaxis (PCA) 151, 153, 156 Peptide 63, 94, 112, 114, 115, 117, 146, 165, 166, 168, 169, 171-174, 188, 194, 196, 198, 212-220, 225, 227, 260, 278 Pharmacological probe 77 Phosphorylation 3, 4, 7, 30, 34, 36, 37, 52, 54, 55, 66, 67, 69, 74, 100, 103, 116, 117, 124, 148-155, 255 Piecemeal degranulation 108, 170, 280 Plasminogen activator 214, 275, 277 Poorly cytokinergic 33, 34 Progenitor 3, 6, 14-25, 40, 125, 127, 128, 133, 135, 136, 146, 152, 187, 257 Prostaglandin 5, 23, 30, 144, 146, 150, 169, 188, 192, 236 Prostaglandin E2 (PGE) 6, 21, 23, 63, 188, 193, 199, 203, 214, 236 Protease 4, 5, 14, 15, 30, 40, 48-50, 107, 146, 169, 171, 188, 190, 192, 194, 196-198, 212-214, 217, 219, 220, 222-224, 226-228, 235-237, 240, 241, 246, 248, 251, 254, 257, 271, 282 Proteinase-activated receptor 2 (PARP 2) 213, 214, 223-225, 259, 276 Protein kinase C (PKC) 8, 37, 77, 116, 117, 130 Proteoglycan 4, 5, 40, 49, 107, 108, 109, 192, 227 Protozoan 166, 171 Puma 4, 36, 51-53

R Raft 7, 34, 35, 38, 91, 92, 94, 95, 97, 102, 117, 118, 127 Recruitment 3, 7, 8, 14, 20-25, 40, 66, 77, 94, 100, 103, 132, 143, 144, 147-151, 154, 155, 164, 165, 169, 171-175, 187, 188, 194, 196-198, 201, 223, 236-238, 245, 246, 248, 249, 251, 257, 271-274, 278, 281 Renin 219-221, 240, 241, 243, 251, 258

293

S Secretion 4, 14, 15, 30, 31, 34, 36, 37, 42, 65, 94, 99, 107, 108, 109, 110, 111, 113-118, 127, 128, 146, 150, 154, 164, 169, 175, 188, 190, 192, 193, 198, 199, 201, 218, 219, 226, 227, 237, 239, 246-249, 254, 256, 257, 259, 270, 273, 274, 278, 280, 281, 282 Secretory granule 14, 15, 34, 107, 190, 219, 227, 278, 280 Sialic acid binding Ig-like lectins (SIGLEC) 147, 150 Signaling 2, 4, 6-8, 16, 22, 24, 31, 32, 34, 36-39, 42, 62, 64-72, 75-77, 79, 80, 91-95, 98-100, 103, 107, 108, 111, 115, 117, 118, 124, 127, 133, 135, 144, 146-148, 152-155, 191, 195, 225, 247, 248, 254, 255 Signal regulatory protein _ (SIRP-_) 145, 147, 151 Signal transduction 7, 30, 31, 33, 36, 37, 102, 116, 117, 144, 151 Skin 16, 19, 20, 49, 53, 80, 128, 129, 134, 136, 139, 153, 154, 156, 163-165, 171-173, 186, 188, 193, 198-200, 202, 203, 212, 213, 217, 219, 224-226, 247, 248, 259, 271, 273-275, 278 SNARE protein 34, 107-114, 116-118 Solid tumor 56, 277 Sphingosine 1-phosphate 5, 32, 67, 123-138, 236, 237 Sphingosine kinase 1 124, 126-130, 134-138 Sphingosine kinase 2 124, 126-131, 134, 135-138 Src family member tyrosine kinase 147, 155 Src homology 2 domain-containing inositol 5- phosphatase (SHIP) 145, 147-151, 153, 154, 256 SHIP1 8, 147 SHIP2 8 Src homology 2 domain-containing tyrosine phosphatase (SHP) 147 SHP-1 8, 145, 147-155

294

MAST CELL BIOLOGY

SHP-2 145, 147, 151-154 Src kinases L Lyn and Fyn 66, 67, 127 Stem cell factor 3-6, 16, 17, 22, 36, 40, 48, 52, 53, 55, 63, 74, 132, 136, 146, 151, 155, 197, 237, 239, 252, 254, 255, 272-274, 282 Store-operated calcium entries 8, 62, 63, 65, 66-72, 74-81 Stromal interaction molecule (STIM) 63 STIM1 8, 62-66, 68-73, 78-81, 130 STIM2 70-72 Survival 3, 4, 6, 20, 24, 25, 29-31, 33-42, 47, 49, 51-56, 124, 146, 151, 173, 187, 190, 196-198, 203, 204, 215, 224, 227, 238-240, 245, 246, 252, 254, 260, 276, 277, 279, 281 Syk 7, 31, 37, 38, 42, 66, 94, 103, 127, 144, 148, 149, 151 Systemic anaphylaxis 5, 110, 133-138, 172, 204

Toll like receptor (TLR) 6, 36, 37, 63, T 144, 164, 167, 170, 172, 173, 192, 193, 246, 247, 254-257 *  < Œ ’ ”^_• Œ ”_ •€ 111, 113, 114, 124, 175 T Tryptase 4, 5, 49, 110, 136, 151, 169, 188, 190, 198, 202, 212-214, 217, 218, 223-225, 227, 237, 239, 241, 246, 248-252, 254, 259, 260, 271, 275-282 Tumor necrosis (TNF)-related T apoptosis-inducing ligand (TRAIL) 4, 50, 53, 54, 146 TRAIL-R 4, 50, 53, 146 Tumor progression 260, 271, 278, 281 T Tyrosine kinase 3, 7, 8, 31, 52, 66, 68, T 91, 94, 100, 103, 116, 127, 144, 147, 148, 189, 272, 281

T

Vaccine adjuvant 176 Vascular cellular adhesion molecule-1 (VCAM)-1 3, 14, 18, 19, 21, 22, 133, 239 Vascular permeability 124, 126, 133, 136-138, 188, 237 Vasoactive intestinal peptide (VIP) 165, 166, 168, 225 Vascular endothelial growth factor (VEGF) 5, 192, 240, 241, 243, 272, 275, 278-280, 282 Vertebrate 162-165, 169, 176 Virus 154, 166, 167, 172, 173, 176, 192, 197, 199, 246, 255, 271

T-bet 3, 14, 19, 20, 79 Termed transient receptor potential T canonical (TRPC) 8, 62-64, 66, 68, 71-73, 78-80 Thrombopoietin (TPO) 19, 20 Tissue 3-6, 14-17, 19, 20, 22-25, 31, 40, 47-49, 52-54, 56, 108, 123-126, 129, 133, 136-138, 146, 152, 154, 163-165, 169-172, 175, 176, 186, 187, 192, 194, 195, 197-199, 201, 202, 204, 212, 214, 216-220, 222, 225-228, 235, 237, 242, 247, 248, 252, 258, 270-272, 274-276, 278, 279, 281, 282

V